AMP-activated protein kinase regulates glycocalyx impairment and macrophage recruitment in response to low shear stress.

Low shear stress (LSS) increases degradation of the endothelial glycocalyx, leading to production of endothelial inflammation and atherosclerosis. However, the underlying mechanisms of how LSS diminishes the endothelial glycocalyx remain unclear. We showed that LSS inactivated AMPK, enhanced Na+-H+ exchanger (NHE)1 activity, and induced glycocalyx degradation. Activation of AMPK prevented LSS-induced NHE1 activity and endothelial glycocalyx impairment. We further identified hyaluronidase 2 (HYAL2) as a mediator of endothelial glycocalyx impairment in HUVECs exposed to LSS. Inactivation of AMPK by LSS up-regulates the activity of HYAL2, which acts downstream of NHE1. We characterized a left common carotid artery partial ligation (PL) model of LSS in C57BL/6 mice. The results showed decreased expression of hyaluronan (HA) in the endothelial glycocalyx and decreased thickness of the endothelial glycocalyx in PL mice. Pharmacological activation of AMPK by ampkinone not only attenuated glycocalyx impairment due to HA degradation but also blocked vascular cell adhesion molecule 1 and intercellular adhesion molecule 1 expression increase and macrophage recruitment in the endothelia of PL mice. Our results revealed that AMPK dephosphorylation induced by LSS activates NHE1 and HYAL2 to promote HA degradation and glycocalyx injury, which may contribute to endothelial inflammatory reaction and macrophage recruitment.-Zhang, J., Kong, X., Wang, Z., Gao, X., Ge, Z., Gu, Y., Ye, P., Chao, Y., Zhu, L., Li, X., Chen, S. AMP-activated protein kinase regulates glycocalyx impairment and macrophage recruitment in response to low shear stress.

Importancia médica del glucocáliz endotelial / Medical significance of endothelial glycocalyx

El glucocáliz endotelial es una capa constituida por glucosaminoglicanos, proteoglicanos y glucoproteínas que cubre al endotelio vascular en su cara luminal. Tiene múltiples funciones: transducción de las fuerzas mecánicas de tensión, regulación de la permeabilidad vascular de líquidos y moléculas y de la activación de la coagulación y de la fibrinólisis, protege de la adhesión de leucocitos y plaquetas al endotelio. En general, el glucocáliz protege a la pared vascular de ataques patogénicos. La lesión del glucocáliz puede ocurrir por fuerzas de tensión anormales, especies reactivas de oxígeno, hipernatremia, hiperglucemia, hipercolesterolemia y moléculas inflamatorias, lo que causa disfunción endotelial, incremento en la permeabilidad, filtración de lipoproteínas al subendotelio, activación de la coagulación e incremento de la adherencia de leucocitos y plaquetas al endotelio vascular. La participación del deterioro del glucocáliz endotelial puede ser importante en la fisiopatología de diversas enfermedades vasculares.

Endothelial glycocalyx is a layer composed by glycosaminoglycans, proteoglycans and glycoproteins attached to the vascular endothelial luminal surface. It has several physiological roles: shear stress mechanotransduction to the endothelial cells, regulation of fluids and macromolecules vascular permeability, of coagulation cascade activation and fibrinolysis, and protects the endothelium from platelets and leukocytes adhesion. In general, glycocalyx protects vascular wall against pathogenic insults. The glycocalyx may be damaged by abnormal shear stress, reactive oxygen species, hypernatremia, hyperglycemia, hypercholesterolemia and inflammatory molecules, resulting in endothelial dysfunction, enhanced vascular permeability, lipoproteins leakage to subendothelial space, activation of plasma coagulation, and increased adherence of platelets and leukocytes to the endothelial cells. Shredding of glycocalyx appears as an important initial step in the pathophysiology of vascular diseases.

Molecular organization of the mucins and glycocalyx underlying mucus transport over mucosal surfaces of the airways.

Mucus, with its burden of inspired particulates and pathogens, is cleared from mucosal surfaces of the airways by cilia beating within the periciliary layer (PCL). The PCL is held to be "watery" and free of mucus by thixotropic-like forces arising from beating cilia. With radii of gyration ~250 nm, however, polymeric mucins should reptate readily into the PCL, so we assessed the glycocalyx for barrier functions. The PCL stained negative for MUC5AC and MUC5B, but it was positive for keratan sulfate (KS), a glycosaminoglycan commonly associated with glycoconjugates. Shotgun proteomics showed KS-rich fractions from mucus containing abundant tethered mucins, MUC1, MUC4, and MUC16, but no proteoglycans. Immuno-histology by light and electron microscopy localized MUC1 to microvilli, MUC4 and MUC20 to cilia, and MUC16 to goblet cells. Electron and atomic force microscopy revealed molecular lengths of 190-1,500 nm for tethered mucins, and a finely textured glycocalyx matrix filling interciliary spaces. Adenoviral particles were excluded from glycocalyx of the microvilli, whereas the smaller adenoassociated virus penetrated, but were trapped within. Hence, tethered mucins organized as a space-filling glycocalyx function as a selective barrier for the PCL, broadening their role in innate lung defense and offering new molecular targets for conventional and gene therapies.

Diazoxide may protect endothelial glycocalyx integrity during coronary artery bypass grafting.

OBJECTIVES: Plasma hyaluronan and syndecan-1 levels represent shedding of the endothelium glycocalyx during ischemia and edema. Diazoxide, a K(ATP)-channel opener, has been shown to decrease myocardial edema during coronary artery bypass grafting (CABG). We evaluated whether diazoxide exerts an impact on plasma hyaluronan and syndecan-1 levels during CABG. DESIGN: Representative blood samples for hyaluronan and syndecan-1, before, during and after surgery, were obtained in 13 out of 16 patients that had a history of stable coronary artery disease undergoing CABG with or without diazoxide. Electron microscopy from biopsies procured from the right atrium in 9 patients was performed to confirm ultrastructural differences among patients before and during CABG. RESULTS: Ultrastructural differences were apparent between individual patients already before operation at base line reflecting differences in the severity of myocardial ischemia and edema. A significant decrease of hyaluronan and syndecan-1 values was observed in patients with diazoxide after surgery (p < 0.04). Significant correlation of plasma hyaluronan and syndecan-1 levels was observed in patients with diazoxide but not in controls (p < 0.005, Spearman rank rho). CONCLUSION: Diazoxide may have an impact on levels of peripheral plasma hyaluronan and syndecan-1 after CABG, suggesting decreased shedding of the endothelial glycocalyx layer.

Shedding of the coronary endothelial glycocalyx: effects of hypoxia/reoxygenation vs ischaemia/reperfusion.

BACKGROUND: Vascular endothelium is covered by a glycocalyx. Damage to the glycocalyx after systemic inflammation or ischaemia/reperfusion contributes to increased vascular permeability and leucocyte adhesion. The underlying mechanisms leading to ischaemia/reperfusion-induced glycocalyx shedding are incompletely understood, in terms of lack of oxygen, absence of flow, or return of oxygen. METHODS: Isolated guinea pig hearts perfused with Krebs-Henseleit buffer at 37°C underwent 20 min of either stopped-flow ischaemia or hypoxic perfusion with subsequent reperfusion/reoxygenation (n = 6 each). Hearts perfused with normoxic buffer served as time controls. Epicardial transudate was collected to assess coronary net fluid filtration, colloid extravasation, and histamine release by mast cells. Syndecan-1 and heparan sulphate were measured in coronary effluent, together with lactate, purines, and the release of mast-cell tryptase ß. Additional hearts were perfusion-fixed to visualize the glycocalyx. RESULTS: Both ischaemia and hypoxia with reperfusion/reoxygenation resulted in significant increases in net fluid filtration (P < 0.05) and release of syndecan-1 and heparan sulphate in coronary effluent. These effects were already seen with the onset of hypoxic perfusion. Histamine was released during hypoxia and reoxygenation and also reperfusion, as was tryptase ß, and high concentrations of adenosine (>1 µmol litre⁻¹, hypoxia group) and inosine (> 7 µmol litre⁻¹, ischaemia group) were measured in effluent (P < 0.05). Damage to the coronary glycocalyx was evident upon electron microscopy. CONCLUSIONS: Both ischaemic and hypoxic hypoxia initiate glycocalyx degradation, promoting an increase in permeability. A contributing mechanism could be purine-mediated degranulation of resident mast cells, with liberated tryptase ß acting as potential 'sheddase'.

Cortisol affects tight junction morphology between pavement cells of rainbow trout gills in single-seeded insert culture.

A primary culture system of rainbow trout gill pavement cells grown on permeable support (single-seeded insert, SSI) was used to examine histological and physiological changes induced by the addition of the corticosteroid hormone cortisol. Pavement cell epithelia were cultured under symmetrical conditions (L15 apical/L15 basolateral) and developed a high transepithelial resistance (TER, 6.84 ± 1.99 kΩ cm(2), mean ± SEM) with a low phenol red diffusion rate (PRD, 0.15 ± 0.03 µmol l(-1)/day). Addition of cortisol to the basolateral compartment increased TER twofold and reduced PRD threefold over a 5-day period. A similar increase in TER could be seen after 24 h apical freshwater (FW) in control cultures. In cortisol-treated cultures FW exposure did not change TER, but PRD increased significantly. Histochemical staining of the cytoskeleton of cells in SSI culture revealed a morphological partitioning into a single mucosal layer of polarized, polygonal cells featuring cortical F-actin rings which were comparable to F-actin rings of epithelial cells on the lamellar and filamental surface, and several unorganized serosal layers of cells with F-actin stress fibers. Addition of cortisol increased cell density by 18% and in the mucosal layer it led to smaller, less polygonal cells with increased height and increased cell contact area. In transmission electron microscopic images two pairs of cytoplasmatic electron-dense structures confining the zonula occludens apically and basally toward the zonula adhaerens were found. Addition of cortisol increased the distance between those paired structures, hence led to deeper tight junctions. The cortisol-induced increase in barrier properties, therefore, involves a structural fortification of the tight junctions which was not generally modified by a short 24-h apical freshwater stress. These results identify cortisol as a regulator of tight junction morphology between pavement cells of euryhaline fish such as the trout.

Structure of capsule surrounding acanthocephalans Corynosoma strumosum in paratenic hosts of three species.

Morphology of capsules surrounding acanthocephalan Corynosoma strumosum in paratenic hosts (sea fishes of three species from the northern part of the Sea of Okhotsk) was studied. A thick layer of glycocalyx is formed on the surface of acanthocephalan's tegument in smelts Osmerus mordax dentex and Hypomesus olidus; the surrounding capsule is formed by fibroblasts and collagen fibers and do not include inflammatory cells. Besides fibroblasts, capsule of the sole Limanda aspera consists also of macrophages, granulocytes, "dark" cells, and once of erythrocytes that indicate obvious inflammatory response of the host's organism to invasion; glycocalyx on the surface of acanthocephalans from the sole is weakly developed. The obtained results allow considering the smelts as the most suitable paratenic hosts and the yellow-finned sole as unsuitable paratenic host for the studied acanthocephalans.

Exfoliative epitheliopathy of bullous keratopathy with breaches in the MUC16 Glycocalyx.

PURPOSE: Expression of cellular adhesion molecules is altered in bullous keratopathy. The hypothesis that epithelial alterations in bullous keratopathy compromise the surface of the cornea and its glycocalyx was tested. METHODS: Studies were performed on eight cases each of pseudophakic bullous keratopathy and healthy corneas. The number of epithelial cell layers was determined with a stereological method of point counting. The minimum distance between points was established by estimates of cell size with variable pressure scanning electron microscopy performed in backscatter mode. The mean number of cell layers with mucin expression was identified by immunohistochemistry with mouse monoclonal antibodies for MUC1 and MUC16. Data were analyzed by Student's t-test if values showed a normal distribution or, alternatively, by the Wilcoxon rank-sum test. RESULTS: Mean numbers of wing cell and superficial cell layers were lower in bullous keratopathy specimens (1.6 vs. 2.0; P < 0.0001) than in controls (1.1 vs. 1.8; P < 0.000001). The number of exfoliated cell layers evident in sections was increased in the bullous keratopathy specimens compared with controls (0.36 vs. 0.03; P < 0.0001). The number of cell layers decorated with antibodies to MUC16 was lower in bullous keratopathy specimens than in controls (0.5 vs. 1.2; P < 0.025). The reduction of layers expressing MUC1 in bullous keratopathy was not statistically significant. CONCLUSIONS: Pseudophakic bullous keratopathy manifests an abnormal corneal ocular surface in which superficial cell layers are exfoliated, leaving breaches in the protective MUC16 glycocalyx. The results provide a morphologic correlate for the surface epithelial abnormalities noted clinically in these patients.

Antithrombin reduces shedding of the endothelial glycocalyx following ischaemia/reperfusion.

AIMS: Antithrombin is an important inhibitor of the coagulation system, additionally exerting specific anti-inflammatory effects on endothelial cells. Healthy vascular endothelium is coated by the endothelial glycocalyx, diminution of which increases capillary permeability, e.g. after ischaemia. Antithrombin is known to infiltrate the glycocalyx, binding to glycosaminoglycans, and to preserve the glycocalyx after application tumour necrosis factor-alpha. We investigated the influence of antithrombin on glycocalyx subjected to ischaemia/reperfusion. METHODS AND RESULTS: Isolated guinea pig hearts were perfused with Krebs-Henseleit buffer (KHB). Antithrombin was applied to achieve physiological levels (1 U/mL) before inducing 20 min of ischaemia (37 degrees C). Hearts were reperfused for 20 min at constant flow (baseline perfusion pressure 70 cmH(2)O) with KHB or KHB plus 2 g% hydroxyethyl starch (130 kDa). Coronary net fluid filtration was assessed directly by measuring transudate formation on the epicardial surface. Post-ischaemic coronary release of syndecan-1 and heparan sulfate was quantified by ELISA. Hearts were perfusion-fixed to visualize the glycocalyx by electron microscopy. Ischaemia/reperfusion caused degradation of the glycocalyx, enhanced coronary perfusion pressure, and increased vascular permeability. Antithrombin significantly reduced post-ischaemic glycocalyx shedding, coronary perfusion pressure, coronary leak, and tissue oedema formation compared to untreated hearts. Additional application of colloid augmented these actions of antithrombin. Electron microscopy revealed a mostly intact glycocalyx after antithrombin treatment. CONCLUSION: Antithrombin preserves the endothelial glycocalyx, sustaining the vascular barrier function and reducing interstitial oedema. The potentiated effect of colloid in these hearts suggests that the prevention of shedding should be of functional benefit also in vivo.

[Expedition glycocalyx. A newly discovered "Great Barrier Reef"]. / Expedition Glykokalyx. Ein neu entdecktes "Great Barrier Reef."

Healthy vascular endothelium is luminally coated by an endothelial glycocalyx, which interacts with the bloodstream and assumes a filter function on the vascular wall. Although this structure was discovered nearly 70 years ago, its physiological importance has been underestimated for a long time. Recent findings indicate that the glycocalyx is, in addition to the endothelial cells themselves, a main constituent part of the vascular barrier. The existence of different colloid osmotic gradients within and beneath this structure has now led to a modification of the Starling equation. In many vascular beds the interstitial space features a protein concentration similar to that of the plasma. The inwardly directed gradient, which retains water and proteins in the vascular system, is generated beneath the glycocalyx by selective protein filtration over this structure. The endothelial glycocalyx, as an additional competent vascular permeability barrier has, therefore, not only a key role for perioperative fluid and protein shifts into the interstitial space, but it seems to be intimately involved in the pathophysiology of diabetes, arteriosclerosis, sepsis and ischemia/reperfusion, especially with respect to associated vascular dysfunctions. The fragile glycocalyx can be destroyed in the course of surgery, trauma, ischemia/reperfusion and sepsis and by inflammatory mediators such as TNF-alpha, causing leukocyte adhesion, platelet aggregation and edema formation. Recent studies have shown that protecting this structure not only maintains the vascular barrier, but constitutes an important component of a rational perioperative fluid therapy.

Characterization of mucosal biofilms on human adenoid tissues.

OBJECTIVES: To demonstrate the presence of mucosal biofilm in adenoid tissue using double staining for visualization of both the bacterial matrix and the bacterial cells. To identify bacterial species present on the surface of the studied adenoids. STUDY DESIGN: Prospective study. METHODS: A total of 39 specimens of adenoidectomy were removed from children with chronic and/or recurrent otitis media. The specimens were prepared for light microscopy using Gram staining, scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). Double staining was performed with CLSM to visualize both the bacteria and the glycocalyx matrix. Nine adenoids on which bacterial biofilms were visualized with CLSM were used for identification of bacterial species by 16S-DNA polymerase chain reaction (PCR) amplification and homology analysis. RESULTS: Of the 39 adenoids investigated, 22 (54%) showed evidence of mucosal biofilms. Gram staining, SEM and CLSM showed the presence of bacterial cells, organized in bacterial microcolonies. CLSM with double staining demonstrated mucosal biofilms by showing the presence of both bacteria and the glycocalyx. The use of 16S-DNA polymerase chain reaction (PCR) amplification and subsequent sequence analyses identified the presence of Corynebacterium argentoratense, Streptococcus salivarius, Micrococcus luteus, and Staphylococcus aureus. CONCLUSIONS: This study demonstrates that adenoid tissue in children with chronic or/and recurrent otitis media contains mucosal biofilms in 54% of the cases. The existence of living bacteria has been demonstrated. Further studies are required to describe the panel of bacteria that can be harbored in the biofilms present in adenoids and the mechanisms involved in the physiopathology of otitis prone children.

Demonstration of bacterial cells and glycocalyx in biofilms on human tonsils.

OBJECTIVES: To demonstrate mucosal biofilms in human tissue by direct visualization of bacteria and glycocalyx using confocal laser scanning microscopy with double fluorescent staining on tonsils and to compare the findings with the results of scanning electron microscopy analysis. DESIGN: Prospective study. SETTING: Tertiary university-based referral center. PATIENTS: Twenty-four tonsils were obtained from children with chronic or recurrent tonsillitis. INTERVENTIONS: Tonsils were prepared for analysis by scanning electronic microscopy and by confocal laser scanning microscopy. MAIN OUTCOME MEASURES: Double fluorescent staining for confocal laser scanning microscopy consisted of propidium iodide staining to detect bacterial cells and fluorescein isothiocyanate concanavalin A staining to detect the glycocalyx matrix. Images were analyzed for characteristic biofilm morphologic features by 3 investigators who evaluated the images independently in a blinded retrospective manner. Consensus of all observers was required to demonstrate the presence of a biofilm in a specimen. RESULTS: Findings from analyses using scanning electronic microscopy suggested the presence of biofilm formations on tonsils by showing bacterial cells in microcolonies. Double-staining technique using confocal laser scanning microscopy showed bacterial cells and the glycocalyx matrix, providing visual evidence for the presence of biofilms on tonsils. CONCLUSION: Using a novel visualization approach in single sections of human mucosal tissue, the presence of biofilms was demonstrated on tonsils in most (17/24 [70.8%]) patients with tonsillitis.

Ultrastructure of the adhesion of bacteria to the epithelial cell membrane of three-day postnatal rat tongue mucosa: a transmission and high-resolution scanning electron microscopic study.

Togue mucosa surface of 3-day postnatal rats was examined under transmission electron microscopy (TEM) and high-resolution scanning electron microscopy (HRSEM). For HRSEM analysis, the specimens were fixed in the same solution for 24 h, postfixed in 2% osmiun tetroxide, critical-point dried and coated with platinum-palladium. For TEM analysis, the specimens were fixed using modified Karnovsky solution and embedded in Spurr resin. The results revealed the presence of numerous microplicae in the membrane surface of keratinized epithelial cells to which groups of bacteria were attached. These bacteria were staphylococcus and coccus organized either in rows or at random, which were visualized in three-dimensional HRSEM images. At high magnification, the TEM images revealed the adhesion of bacteria to the cell membrane through numerous filamentous structures comprising the glycocalyx. The fine fibrillar structures rising from each bacterium and from cell membrane were clearly seen. These characteristics on bacteria structure may be used for future control or prevention of bacterial diseases and for installation of the oral native flora.

Ultrastructure of the adhesion of bacteria to the epithelial cell membrane of three-day postnatal rat tongue mucosa: a transmission and high-resolution scanning electron microscopic study

Togue mucosa surface of 3-day postnatal rats was examined under transmission electron microscopy (TEM) and high-resolution scanning electron microscopy (HRSEM). For HRSEM analysis, the specimens were fixed in the same solution for 24 h, postfixed in 2 percent osmiun tetroxide, critical-point dried and coated with platinum-palladium. For TEM analysis, the specimens were fixed using modified Karnovsky solution and embedded in Spurr resin. The results revealed the presence of numerous microplicae in the membrane surface of keratinized epithelial cells to which groups of bacteria were attached. These bacteria were staphylococcus and coccus organized either in rows or at random, which were visualized in three-dimensional HRSEM images. At high magnification, the TEM images revealed the adhesion of bacteria to the cell membrane through numerous filamentous structures comprising the glycocalyx. The fine fibrillar structures rising from each bacterium and from cell membrane were clearly seen. These characteristics on bacteria structure may be used for future control or prevention of bacterial diseases and for installation of the oral native flora.

A superfície lingual de ratos de três dias de idade foi examinada em microscópia eletrônica de transmissão (MET) e em microscópia eletrônica varredura de alta resolução (MEVAR). Para o método de MEVAR, os espécimes foram fixados na mesma solução por 24 h, pós fixados em solução de tetróxido de ósmio a 2 por cento, secos em ponto crítico e cobertos com platina- paládio. Para análise em MET, os espécimes foram fixados utilizando-se solução de Karnovsky modificada e emblocadas em resina Spurr. Os resultados mostraram a presença de numerosas micropregas na membrana superficial das células epiteliais queratinizadas, nas quais estavam aderidos grupos de bactérias. Estas bactérias eram estafilococos e cocos, organizados em fileiras ou a esmo, e puderam ser observadas em imagens tri-dimensionais em MEVAR. Em maiores aumentos, as imagens em MET revelaram a adesão de bactérias nas células por meio de numerosas estruturas filamentares compondo o glicocálice. As delicadas estruturas filamentares na periferia das bactérias e das células foram nitidamente identificadas. Estas características da estrutura bacteriana podem ser utilizadas, no futuro, para controle e prevenção de doenças bacteriana, bem como para a instalação da flora oral nativa.

Bacterial internalization in periodontitis.

BACKGROUND: Bacterial invasion of host epithelial cells plays an important role in the pathogenesis of periodontitis; however, the exact mechanism of the invasion has not been investigated. METHODS: Pocket epithelium biopsies in periodontitis were analysed via scanning and transmission electron microscopy using ultra-histochemical staining with ruthenium red for glycocalyx visualization. RESULTS: We demonstrated that oral bacteria adhered via fimbriae-mediated adhesion only. The bacterial internalization in periodontitis was marked by the hallmark of the fimbriae-induced zipper mechanism--the phagocytic cup formation--but we found no sign of the trigger mechanism of internalization. In addition, we frequently observed apoptosis in the phagocytizing epithelial cells. CONCLUSION: Fimbriae-mediated adhesion is a prerequisite for bacterial invasion in periodontitis. This occurs by the fimbriae-induced zipper mechanism of internalization. As internalization induces apoptosis, the subsequent exfoliation might play a significant role in the clearance of periodontal pathogens.

Bacterial biofilms in surgical specimens of patients with chronic rhinosinusitis.

OBJECTIVES: Biofilms are bacterial pathogens that organize in several chronic and recalcitrant infectious processes. We hypothesize that biofilms play a role in chronic rhinosinusitis (CRS). Our goal is to demonstrate biofilms in mucosal specimens of patients undergoing surgery for CRS. STUDY DESIGN: A prospective study of the presence of biofilms in patients undergoing endoscopic sinus surgery for CRS compared with control patients without CRS. METHODS: There were a total of 30 subjects and 4 controls enrolled. The samples of 24 subjects and 4 controls were cultured and then prepared using standard methods for scanning electron microscopy (SEM). The remaining six subjects' samples were treated using advanced cryofixation methods as preparation to preserve structure for SEM and transmission electron microscopy (TEM). RESULTS: Using strict SEM morphologic criteria, 24 (80%) of the 30 patients were found to have micrographic evidence of biofilms. All controls had healthy appearing cilia and goblet cells without biofilms. The six cryofixation samples showed biofilm structures on SEM micrographs that were correlated with bacterial structures seen at the mucosal surface on the corresponding TEM cross sections. Bacterial cultures were positive on all patients. CONCLUSIONS: Biofilms were demonstrated to be present in patients undergoing surgery for CRS; none of the patients without CRS had any evidence of biofilms. Although SEM is capable of demonstrating the biofilms' three-dimensional structure, glycocalyx, and water channels, it cannot clearly demonstrate the presence of bacteria within the biofilm. We were able to demonstrate evidence of bacteria in the biofilms on the subjects tested using TEM.

Carcinoembryonic antigen (CEA) and CEA-related cell adhesion molecule 1 (CEACAM1), apically expressed on human colonic M cells, are potential receptors for microbial adhesion.

In the human gut mucosa, specialized M cells deliver intact foreign macromolecules and commensal bacteria from the lumen to organized mucosal lymphoid tissues triggering immune responses. M cells are also major sites of adhesion and invasion for enteric pathogens. The molecular features of M cell apical surfaces that promote microbial normal attachment are still largely unknown. We have demonstrated previously that in the human colonic epithelium, carcinoembryonic antigen (CEA) and CEA-related cell adhesion molecule 1 (CEACAM1) are integral components of the apical glycocalyx which participate in epithelial-microbial interactions. In this study, based on the reactivity of specific monoclonal antibodies and on immunoelectron microscopy, we show that M cells of human colonic solitary lymphoid follicles express CEA and CEACAM1 on the apical surface. Recently these highly glycosylated molecules have been characterized as protein receptors for different bacteria. This leads us to propose a role for CEA and CEACAM1 in the adherence of enteric bacteria to the apical membrane of colonic M cells. We also hypothesize that, unlike colonic enterocytes, M cells lack the defense mechanism that eliminates CEA and CEACAM1 upon microbial binding and which is based on vesiculation of microvillus plasma membrane.

Morphology of the midgut trunk in the penaeid shrimp, Sicyonia ingentis, highlighting novel nuclear pore particles and fixed hemocytes.

The morphology of the midgut trunk (MGT) in the penaeid shrimp Sicyonia ingentis was examined by light and scanning and transmission electron microscopy. Although the function of the MGT is poorly understood, it is not involved with the digestion and absorption of nutrients, and it appears to be the surface of a shrimp least protected from penetration by potential pathogens. As described for other decapod crustaceans, the MGT in shrimp is composed of a simple columnar epithelium separated from a layer of connective tissue by a thick basal lamina. Beneath the basal lamina is a previously unreported layer of hemocytes, exclusively of the granulocyte variety, embedded in a matrix continuous with the basal lamina and extending into the connective tissue. This layer was observed in four other species of penaeid shrimp. Granulocytes in circulation can phagocytose and encapsulate foreign material and the granules contain antibacterial molecules, lysosomal enzymes, and prophenoloxidase. We suggest that the granulocytes associated with the basal lamina have matured at this site and are well positioned to fight potential pathogens that have penetrated the epithelial layer of the MGT. A second observation is the presence of clusters of cylinders bound to the nuclear pores of the epithelial cells. The possibility that these clusters are viruses, organelles, or abnormal organelles induced by disease or toxic materials is discussed. These unique particles were observed in S. ingentis but none of the other penaeid shrimp we examined.

Stroma-mediated granulocyte-macrophage colony-stimulating factor (GM-CSF) control of myelopoiesis: spatial organisation of intercellular interactions.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is one of the major cytokines involved in control of haemopoiesis both in bone marrow and in extramedullar sites. Its biological activity depends upon the composition and physicochemical properties of the microenvironment provided by the supporting stroma. GM-CSF activity is modulated and controlled by the stromal heparan-sulphate proteoglycans, but their optimal interaction occurs only at low pH. We questioned whether the microenvironment organisation of the interface between stroma and haemopoietic cells provides such conditions. We studied myeloid progenitor proliferation in contact with bone marrow-derived and extramedullar stromas using electron microscopy and selective labelling of pericellular components. We present evidence that, upon interaction, the two cell types reorganise their interface both in shape and molecular composition. Haemopoietic cells extend projections that considerably increase the area of intercellular contact, and stromal cells form lamellipodia and carry out a redistribution of membrane-associated sialylated glycoconjugates and proteoglycans. Such rearrangements lead to extensive capping of negatively charged molecules at the interface between the supporting stroma and the haemopoietic cells, leading potentially to a local decrease in pH. Our results indicate that the distribution of negative charges at the cellular interface may be responsible for the selectivity of cell response to GM-CSF.

Apical barriers to airway epithelial cell gene transfer with amphotropic retroviral vectors.

Gene transfer to airway epithelia with amphotropic pseudotyped retroviral vectors is inefficient following apical vector application. To better understand this inefficiency, we localized the expression of Pit2, the amphotropic receptor, in polarized human airway epithelia. Pit2 was expressed on both the apical and basolateral surfaces of the cells, suggesting that factors other than receptor abundance may limit apical gene transfer efficiency. Binding studies performed with radiolabeled amphotropic MuLV suggested that the apically applied virus binds to Pit2. Hypothetical barriers to retroviral gene transfer include the apical glycocalyx and other secreted products of epithelia. In this study, we demonstrated that sialic acid, keratan sulfate and collagen type V are present on the apical surface of well-differentiated human airway epithelia. While enzyme treatment reduced the abundance of these components, the treatment also decreased the transepithelial resistance to approximately 35% of the controls, suggesting that the epithelial integrity was impaired. To attain an airway epithelial culture with a modified apical surface and intact epithelial integrity, we utilized 100 mM 2-deoxy-D-glucose, a glycosylation inhibitor, to prevent the glycocalyx from reforming following enzyme treatment. This approach allowed the resistance, but not the apical glycocalyx to recover. Despite this physical modification of the cell surface, the amphotropic retroviral vector failed to transduce airway epithelia following apical application. These results suggest that factors other than apical receptor abundance and the glycocalyx inhibit amphotropic retroviral gene transfer in human airway epithelia.