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PubMed1988

Schistosoma mansoni: ultrastructural demonstration of a miracidial glycocalyx that cross-reacts with antibodies raised against the cercarial glycocalyx.

Cercariae are covered by a glycocalyx that is highly antigenic. Here, we have examined the surface of miracidia for a similar structure. The miracidia are covered by epithelial plates and syncytial ridges. By transmission electron microscopy, the plates and ridges were covered by a 0.5-micron-thick glycocalyx composed of a mesh of 9- to 10-nm fibrils that were stained by ruthenium red delivered in the aldehydes or ferrocyanide-reduced osmium tetroxide. Rabbit antibodies prepared against phenol extracted and chromatographed cercarial glycocalyx were detected by immunoelectron microscopy with secondary antibodies conjugated to horseradish peroxidase. Reaction product bound to both the miracidial and cercarial glycocalyx. In addition, the outer leaflets of the cercarial tegumental membrane and membranes of the miracidial surface structures, including plates, ridges, terebratorium, and sensory papillae, had reaction product. Controls incubated with nonspecific rabbit serum had no reaction product. By indirect immunofluorescence, antibodies against the cercarial glycocalyx stained both plates and ridges. As the miracidia transformed to sporocysts, the glycocalyx remained associated with the plates as they were sloughed. These studies demonstrate that miracidia possess a glycocalyx similar in structure and antigenicity to the cercarial glycocalyx.

Animals↗
PubMed1998

Ischemic preconditioning and superoxide dismutase protect against endothelial dysfunction and endothelium glycocalyx disruption in the postischemic guinea-pig hearts.

UNLABELLED: The effect of ischemic preconditioning and superoxide dismutase (SOD) on endothelial glycocalyx and endothelium-dependent vasodilation in the postischemic isolated guinea-pig hearts was examined. Seven groups of hearts were used: group 1 underwent sham aerobic perfusion; group 2 was subjected to 40 min global ischemia without reperfusion; group 3, 40 min ischemia followed by 40 min reperfusion; group 4 was preconditioned with three cycles of 5 min global ischemia followed by 5 min of reperfusion (IPC), prior to 40 min ischemia; group 5 was subjected to IPC prior to standard ischemia/ reperfusion; group 6 underwent standard ischemia/reperfusion and SOD infusion (150 U/ml) was begun 5 min before 40 min ischemia and continued during the initial 5 min of the reperfusion period; group 7 was subjected to 80 min aerobic perfusion with NO-synthase inhibitor, L-NAME, to produce a model of endothelial dysfunction independent from the ischemia/reperfusion. Coronary flow responses to acetylcholine (ACh) and sodium nitroprusside (SNP) were used as measures of endothelium-dependent and endothelium-independent vascular function, respectively. Reduction in coronary flow caused by NO-synthase inhibitor, L-NAME, served as a measure of a basal endothelium-dependent vasodilator tone. After completion of each experimental protocol, the hearts were stained with ruthenium red or lanthanum chloride for electron microscopy evaluation of the endothelial glycocalyx. While ischemia led only to a slightly flocculent appearance of the glycocalyx, in ischemia/reperfused hearts the glycocalyx was disrupted, suggesting that it is the reperfusion injury which leads to the glycocalyx injury. Moreover, the coronary flow responses to ACh and L-NAME were impaired, while the responses to SNP were unchanged in the ischemia/reperfused hearts. The disruption of the glycocalyx and the deterioration of ACh and L-NAME responses was prevented by IPC. In addition, the alterations in the glycocalyx and the impairment of ACh responses were prevented by SOD. The glycocalyx appeared to be not changed in the hearts subjected to 80 min aerobic perfusion with L-NAME. IN CONCLUSION: (1) the impairment of the endothelium-dependent coronary vasodilation is paralleled by the endothelial glycocalyx disruption in the postischemic guinea-pig hearts; (2) both these changes are prevented by SOD, suggesting the role of free radicals in the mechanism of their development; (3) both changes are prevented by IPC. We hypothesize, therefore, that alterations in the glycocalyx contribute to the mechanism of the endothelial dysfunction in the postischemic hearts.

Acetylcholine↗
PubMedDate not supplied

Ultrastructural demonstration of endothelial glycocalyx disruption in the reperfused rat heart. Involvement of oxygen free radicals.

To determine the effect of post-ischaemic reperfusion on the ultrastructure of the endothelial glycocalyx and the role of oxygen free radicals, isolated working rat hearts were subjected to 20 min ischaemia followed by 3 or 30 min of reperfusion. Ruthenium red and lanthanum chloride were used to delineate the endothelial glycocalyx, and histochemical manganese/diaminobenzidine (Mn+2/DAB) or iron/diaminobenzidine (Fe+2/DAB) techniques were applied to visualize superoxide and hydrogen peroxide in myocardial capillaries. We found that ischaemia alone led to only a slightly flocculent appearance of the glycocalyx and its disruption was not observed until the onset of reperfusion. Prolongation of reperfusion to 30 min had no further effect on the ultrastructure of the glycocalyx. The ultrastructure of endothelial cells was normal. The disruption of the glycocalyx correlated in time and place with the appearance of Mn+2/DAB and Fe+2/DAB reaction products on the luminal surface of endothelial cells. Treatment with 5 mM N-(2-mercaptopropionyl)-glycine (MPG), an .OH radical scavenger, starting before ischaemia prevented the disruption of the glycocalyx, while 100 mM 3-morpholinosydnonimine (SIN-1), capable of generating both NO and -O2 simultaneously when applied at the time of reperfusion, increased the mean density of capillaries positively stained with Mn+2/DAB and Fe+2/DAB, and caused substantial disruption of the glycocalyx and damage to endothelial cells, which was not prevented by MPG. Our results suggest that the onset of reperfusion is critical for injury to the endothelial glycocalyx. Most probably the hydroxyl radical derived from the Fenton reaction is responsible for this injury. Peroxynitrite and/or nitric dioxide, if present upon reperfusion, may also account for damage of the endothelial glycocalyx.

Animals↗
PubMed1994

Staphylococcal glycocalyx activates macrophage prostaglandin E2 and interleukin 1 production and modulates tumor necrosis factor alpha and nitric oxide production.

We have examined the effect of staphylococcal glycocalyces on the ability of murine peritoneal macrophages to produce prostaglandin E2 (PGE2) and the inflammatory cytokines interleukin 1 (IL-1) and tumor necrosis factor alpha (TNF-alpha) and to generate nitric oxide. Glycocalyx partially purified under endotoxin-free conditions from defined liquid medium cultures of Staphylococcus lugdunensis or Staphylococcus epidermidis was a strong stimulator of PGE2 and IL-1 production. The addition of 10 to 100 micrograms of glycocalyx per ml induced levels of IL-1 and PGE2 production similar to that induced by 0.1 to 1 micrograms of Escherichia coli lipopolysaccharide (LPS) per ml. In contrast, glycocalyx induced ninefold less TNF-alpha and three- to fourfold less nitrite than LPS. A modulatory effect was suggested by the observation that the amount of TNF-alpha and nitrite generated remained constant whether the macrophages were stimulated with 10 or 100 micrograms of glycocalyx per ml. A selective modulation of macrophage activation was confirmed by the demonstration that costimulation of macrophages with both glycocalyx and LPS resulted in a reduction in TNF-alpha and nitrite generation relative to stimulation with LPS alone even though costimulation had no effect on PGE2 production and increased IL-1 production. Involvement of PGE2 in this modulatory effect was suggested by the ability of indomethacin to augment glycocalyx-stimulated TNF-alpha production and to reverse the inhibitory effect of glycocalyx on LPS induction of TNF-alpha production. However, the inability of indomethacin to reverse the inhibitory effect of glycocalyx on LPS-induced nitric oxide generation suggests that the selective modulation of macrophage function by glycocalyx may be more complex than increased sensitivity to PGE2 feedback inhibition.

Animals↗
PubMed1986

Electron microscopic investigation of the bladder urothelium and glycocalyx in patients with interstitial cystitis.

The electron microscopic appearance of the bladder urothelium and glycocalyx was investigated in ten patients with well defined interstitial cystitis and compared to the findings in ten control patients with stress incontinence as the only symptom. Ruthenium red, a polycationic dye which binds specifically to cell surface acid polysaccharides, was used to demonstrate the glycocalyx. In cases of interstitial cystitis two types of luminal cell were observed, each possessing a distinct surface glycocalyx. One type of cell possessed numerous plaques of asymmetric unit membrane associated with a relatively thin glycocalyx. The second type of cell was characterised by numerous microvilli and a relatively thick glycocalyx. In control material each type of cell and its associated glycocalyx was identified with similar frequency. Our study concludes that there are no differences in the morphologic appearances of the glycocalyx and of urothelial cells in patients with interstitial cystitis when compared with controls. Hence, the hypothesis that an important pathogenic factor in interstitial cystitis is a defective glycocalyx associated with a permeable urothelium, has not been supported.

Adult↗
PubMed1989

The role of glycocalyx in surface phagocytosis of Bacteroides spp., in the presence and absence of clindamycin.

The influence of isolated glycocalyx from Bacteroides thetaiotaomicron and B. fragilis on surface phagocytosis of clindamycin-treated and -untreated homologous and heterologous species was studied. When homologous or heterologous isolated glycocalyx was added to clindamycin-treated B. thetaiotaomicron or B. fragilis before incubation with PMNL, phagocytosis was reduced to levels observed in the untreated control bacteria, but addition of glycocalyx to untreated control strains showed no reduction of phagocytosis. When isolated bacteroides-glycocalyx was added to Staphylococcus aureus or S. epidermidis, phagocytosis of both clindamycin-treated and -untreated bacteria was significantly reduced. The isolated glycocalyx preparations were analysed by thin layer and gas-liquid chromatography; these preparations were free of lipopolysaccharides. The isolated glycocalyx did not affect PMNL viability. Our findings suggest that the glycocalyx is an important virulence factor because it impairs phagocytosis of Bacteroides spp. by PMNL. Clindamycin may enhance opsonophagocytosis of bacteroides by altering the glycocalyx.

Bacteroides↗
PubMed1989

Synthesis of glycocalyx and associated structures in vestibular sensory cells.

The ultrastructure of the glycocalyx with special reference to the synthesizing process was studied in the guinea pig vestibular sensory cells using the tannic acid staining technique. The glycocalyx emerged from the outer layer of the plasma membrane covering the entire length of the cilia. This glycocalyx also interconnected the ciliary structures tightly, such that a structural continuity was established between actin-membrane links and the glycocalyx. Interconnections between the actin filaments themselves were also noticed in the stereocilia as well as interconnections between individual actin filaments and the plasma membrane. These findings indicate that the glycocalyx and the ciliary interconnections may be closely related to the sensory hair transduction system. In the cellular cytoplasm, vesicles seemingly related to the synthesis of the glycocalyx were observed. These coated vesicles, which were synthesized by the Golgi complex and endoplasmic reticulum, interacted with the plasma membrane forming a coated pit. The lysosomal-like bodies also observed in the cell were closely related to the glycocalyx as well. Thus the glycocalyx seems to be synthesized by the endoplasmic reticulum and Golgi complexes and transferred through the coated vesicles or lysosomal-like bodies to the apical plasma membrane.

Animals↗
PubMed1983

The use of specific antibodies to demonstrate the glycocalyx and spatial relationships of a K99-, F41- enterotoxigenic strain of Escherichia coli colonizing the ileum of colostrum-deprived calves.

Electron microscopy was used to study the interaction between the glycocalyx of enterotoxigenic Escherichia coli strain 210 (09:K30+;K99-;F41-:H-) and the glycocalyx of epithelial cells in then ileum of experimentally infected newborn colostrum-deprived calves. Fixation of tissues in anti-K30 antibody and ruthenium red was used to stabilize the bacterial glycocalyx so that the spatial relationship between the bacteria and the intestinal epithelial cells could be characterized. When strain 210 was grown in vitro and reacted with anti-K30 antibody prior to staining with ruthenium red, the extensive glycocalyx could be clearly visualized surrounding the bacterial cells. By negative staining, an unidentified pilus was also seen. Sections of ileum from infected calves, which were not fixed in antibody nor stained with ruthenium red, revealed attached bacteria which were surrounded by an electron-translucent zone and no visible bacterial glycocalyx. When ruthenium red staining was used, the bacterial glycocalyx partially collapsed during the dehydration steps of fixation, but could be seen as either a fibrous capsule or an electron-dense accretion on the bacterial cell surface. When ileal tissue was reacted for one hour in anti-K30 antibody before staining with ruthenium red, the bacterial glycocalyx was seen as a discrete electron-dense structure up to 1.0microm thick which was in intimate contact with the glycocalyx of the epithelial cells. The importance of the bacterial exopolysaccharide to microcolony formation on the villi could be clearly visualized.

Animals↗
PubMed2001

An electrochemical model of the transport of charged molecules through the capillary glycocalyx.

An electrochemical theory of the glycocalyx surface layer on capillary endothelial cells is developed as a model to study the electrochemical dynamics of anionic molecular transport within capillaries. Combining a constitutive relationship for electrochemical transport, derived from Fick's and Ohm's laws, with the conservation of mass and Gauss's law from electrostatics, a system of three nonlinear, coupled, second-order, partial, integro-differential equations is obtained for the concentrations of the diffusing anionic molecules and the cations and anions in the blood. With the exception of small departures from electroneutrality that arise locally near the apical region of the glycocalyx, the model assumes that cations in the blood counterbalance the fixed negative charges bound to the macromolecular matrix of the glycocalyx in equilibrium. In the presence of anionic molecular tracers injected into the capillary lumen, the model predicts the size- and charge-dependent electrophoretic mobility of ions and tracers within the layer. In particular, the model predicts that anionic molecules are excluded from the glycocalyx at equilibrium and that the extent of this exclusion, which increases with increasing tracer and/or glycocalyx electronegativity, is a fundamental determinant of anionic molecular transport through the layer. The model equations were integrated numerically using a Crank-Nicolson finite-difference scheme and Newton-Raphson iteration. When the concentration of the anionic molecular tracer is small compared with the concentration of ions in the blood, a linearized version of the model can be obtained and solved as an eigenvalue problem. The results of the linear and nonlinear models were found to be in good agreement for this physiologically important case. Furthermore, if the fixed-charge density of the glycocalyx is of the order of the concentration of ions in the blood, or larger, or if the magnitude of the anionic molecular valence is large, a closed-form asymptotic solution for the diffusion time can be obtained from the eigenvalue problem that compares favorably with the numerical solution. In either case, if leakage of anionic molecules out of the capillary occurs, diffusion time is seen to vary exponentially with anionic valence and in inverse proportion to the steady-state anionic tracer concentration in the layer relative to the lumen. These findings suggest several methods for obtaining an estimate of the glycocalyx fixed-charge density in vivo.

Algorithms↗
PubMed1998

Adhesion-related glycocalyx study: quantitative approach with imaging-spectrum in the energy filtering transmission electron microscope (EFTEM).

Large polysaccharide molecules composing the glycocalyx have been shown to prevent cell adhesion. However, this process was not observed microscopically. Terbium labeling, combined with a new quantitative imaging method based on electron energy loss spectroscopy, allowed specific glycocalyx staining with excellent contrast. Image analysis enabled us to compare glycocalyx structure in free membrane areas and contacts between monocytic cells and bound erythrocytes. Apparent glycocalyx thickness, in contact areas, was half of the sum of glycocalyx thicknesses in free areas without label density increase. Ultrastructural immunogold localization of CD43 molecules, a major component of glycocalyx, was also demonstrated to be excluded from contact areas during adhesion. Thus, both approaches strongly suggest that some glycocalyx elements must exit from contact to allow binding of adhesion molecules.

Antigens, CD↗
PubMed1999

Permeation of the luminal capillary glycocalyx is determined by hyaluronan.

The endothelial cell glycocalyx influences blood flow and presents a selective barrier to movement of macromolecules from plasma to the endothelial surface. In the hamster cremaster microcirculation, FITC-labeled Dextran 70 and larger molecules are excluded from a region extending almost 0.5 micrometer from the endothelial surface into the lumen. Red blood cells under normal flow conditions are excluded from a region extending even farther into the lumen. Examination of cultured endothelial cells has shown that the glycocalyx contains hyaluronan, a glycosaminoglycan which is known to create matrices with molecular sieving properties. To test the hypothesis that hyaluronan might be involved in establishing the permeation properties of the apical surface glycocalyx in vivo, hamster microvessels in the cremaster muscle were visualized using video microscopy. After infusion of one of several FITC-dextrans (70, 145, 580, and 2,000 kDa) via a femoral cannula, microvessels were observed with bright-field and fluorescence microscopy to obtain estimates of the anatomic diameters and the widths of fluorescent dextran columns and of red blood cell columns (means +/- SE). The widths of the red blood cell and dextran exclusion zones were calculated as one-half the difference between the bright-field anatomic diameter and the width of the red blood cell column or dextran column. After 1 h of treatment with active Streptomyces hyaluronidase, there was a significant increase in access of 70- and 145-kDa FITC-dextrans to the space bounded by the apical glycocalyx, but no increase in access of the red blood cells or in the anatomic diameter in capillaries, arterioles, and venules. Hyaluronidase had no effect on access of FITC-Dextrans 580 and 2,000. Infusion of a mixture of hyaluronan and chondroitin sulfate after enzyme treatment reconstituted the glycocalyx, although treatment with either molecule separately had no effect. These results suggest that cell surface hyaluronan plays a role in regulating or establishing permeation of the apical glycocalyx to macromolecules. This finding and our prior observations suggest that hyaluronan and other glycoconjugates are required for assembly of the matrix on the endothelial surface. We hypothesize that hyaluronidase creates a more open matrix, enabling smaller dextran molecules to penetrate deeper into the glycocalyx.

Animals↗
PubMed2000

TNF-alpha increases entry of macromolecules into luminal endothelial cell glycocalyx.

The endothelial luminal glycocalyx has been largely ignored as a target in vascular pathophysiology even though it occupies a key location. As a model of the inflammatory response, we tested the hypothesis that tumor necrosis factor-alpha (TNF-alpha) can alter the properties of the endothelial apical glycocalyx. In the intact hamster cremaster microcirculation, fluorescein isothiocyanate (FITC)-labeled Dextrans 70, 580, and 2,000 kDa are excluded from a region extending from the endothelial surface almost 0.5 micrometer into the lumen. This exclusion zone defines the boundaries of the glycocalyx. Red blood cells (RBC) under normal flow conditions are excluded from a region extending even farther into the lumen. The cremaster microcirculation was pretreated with topical or intrascrotal applications of TNF-alpha. After infusion of FITC-dextran, FITC-albumin, or FITC-immunoglubulin G (IgG) via a femoral cannula, microvessels were observed with bright-field and fluorescence microscopy to obtain estimates of the anatomic diameters and the widths of fluorescent tracer columns and of the RBC columns (means +/- SE). After 2 h of intrascrotal TNF-alpha exposure, there was a significant increase in access of FITC-Dextrans 70 and 580 to the space bounded by the apical glycocalyx in arterioles, capillaries, and venules, but no significant change in access of FITC-Dextran 2,000. The effects of TNF-alpha could be observed as early as 20 min after the onset of topical application. TNF-alpha treatment also significantly increased the penetration rate of FITC-Dextran 40, FITC-albumin, and FITC-IgG into the glycocalyx and caused a significant increase in the intraluminal volume occupied by flowing RBC. White blood cell adhesion increased during TNF-alpha application, and we used the selectin antagonist fucoidan to attenuate leukocyte adhesion during TNF-alpha stimulation. This did not inhibit the TNF-alpha-mediated increase in permeation of the glycocalyx. These results show that proinflammatory cytokines can cause disruption of the endothelial apical glycocalyx, leading to an increased macromolecular permeation in the absence of an increase in leukocyte recruitment.

Administration, Topical↗
PubMed2001

Elevated capillary tube hematocrit reflects degradation of endothelial cell glycocalyx by oxidized LDL.

Proteoglycans and plasma proteins bound to the endothelial cell glycocalyx are essential for vascular function, but at the same time, they lower capillary tube hematocrit by reducing capillary volume available to flowing blood. Because oxidized low-density lipoproteins (oxLDL) reduce the effective thickness of the glycocalyx (Vink H, Constantinescu AA, and Spaan JAE. Circulation 101: 1500-1502, 2000), we designed the present study to determine whether this is caused by pathological degradation of glycocalyx constituents or increased glycocalyx deformation by elevated shear forces of flowing blood. Capillaries from the right cremaster muscle of 24 hamsters were examined by using intravital microscopy after systemic administration of normal LDL (n = 4), moderate oxLDL (6-h oxidation with CuSO(4), n = 7), severe oxLDL (18-h oxidation, n = 5), and moderate oxLDL plus superoxide dismutase (SOD) and catalase (n = 8). Capillary tube hematocrit increased from 0.16 +/- 0.03 to 0.37 +/- 0.05 and from 0.15 +/- 0.01 to 0.31 +/- 0.03 after moderate oxLDL and severe oxLDL, respectively. These changes were paralleled by increases in red blood cell flux from 8.7 +/- 1.9 to 13.8 +/- 3 and from 10.7 +/- 2.1 to 16.3 +/- 3.2 cells/s after moderate oxLDL and severe oxLDL, respectively, in the absence of changes in anatomic capillary diameter. Red blood cell velocity, as a measure for the shear forces on the glycocalyx, was not affected by oxLDL, whereas tissue pretreatment with SOD and catalase completely abolished the effects of oxLDL on glycocalyx thickness, capillary hematocrit, and red blood cell flux. We conclude that elevation of capillary tube hematocrit by oxLDL reflects degradation of the endothelial glycocalyx by oxygen-derived free radicals.

Animals↗
PubMed1984

Alterations in epithelial glycocalyx of rabbit uteri during early pseudopregnancy and pregnancy, and following ovariectomy.

Pseudopregnant, pregnant, and ovariectomized rabbits were utilized to study hormonal mediation of uterine epithelial surface negativity and glycocalyx morphology, and to seek local effects of blastocysts at sites of implantatioN. A loss of surface negativity [polycationic ferritin (PCF) binding] by day 6 of pregnancy or pseudopregnancy was noted, accompanied by alterations in epithelial glycocalyx. Uteri from estrous animals, or ovariectomized animals receiving oil or estradiol injections, bound PCF and exhibited a "globular" glycocalyx. Uteri from day 6 pseudopregnant or pregnant animals, or ovariectomized animals receiving progesterone injections, did not bind PCF or exhibit a globular glycocalyx. Both PCF binding and the globular character of the epithelial glycocalyx were sensitive to neuraminidase and trypsin treatment, suggesting sialoglycoprotein contribution to surface negativity. Implanting blastocysts had no detectable local effect on surface negativity, but did induce local reduction of epithelial glycocalyx at sites of implantation. Results of this study suggest that uterine epithelial glycocalyx alterations during the preimplantation period reflect a general response to progesterone stimulation, primarily qualitative in nature, related to the acquisition of receptivity to ovo-implantation.

Animals↗
PubMed1988

Schistosoma mansoni: development of the cercarial glycocalyx.

The development of the cercarial glycocalyx of Schistosoma mansoni was studied by transmission electron microscopy and immunofluorescence light microscopy employing antibodies raised against extracted and chromatographed glycocalyx. By electron microscopy, cercariae present in the brood chamber of daughter sporocysts were surrounded by an electron-dense granular and fibrillar matrix. This material appeared structurally distinct from the glycocalyx which was coarsely fibrillar and located only on the surface of organisms that had developed a final tegument. The thickness of the glycocalyx apparently increased with the maturation of the tegument, since teguments that had many spines also had the thickest glycocalyx. Immunofluorescent staining of frozen sections of infected snail hepatopancreas showed that glycocalyx antigens were present on the surface of the cercariae and not in the matrix within the brood chamber or in snail tissues. Immunofluorescent staining of isolated larval cercariae showed staining of some but not all parasites with partially elongated tails. These studies suggest that the glycocalyx develops late in cercarial development (late in Stage 6 or in Stage 7 of Cheng and Bier), is made by the cercariae themselves, and is not a product of either the sporocyst wall cells or snail hepatopancreas.

Animals↗
PubMed1991

Paragonimus ohirai: immunobiochemical characterization on the tegumental glycocalyx of excysted juvenile recognized by a monoclonal antibody.

The tegumental glycocalyx of excysted juvenile (EJ) of Paragonimus ohirai was immunobiochemically characterized using a monoclonal antibody (MS-Mab). HPLC gel filtration showed that the antigens detected by two-site ELISA had a molecular weight of greater than or equal to 2 x 10(6) Da (dextran marker). On reduced SDS-PAGE, the glycocalyx antigen retained in the stacking gel was cleaved into several much smaller antigens after pronase treatment. The antigenic activity of the glycocalyx was stable in two-site ELISA to heat and acid treatments, but sensitive to alkali, periodate, base/borohydride, and pronase treatments. Precipitin formation in immunodouble diffusion between MS-Mab and EJ crude antigen was inhibited only by two monosaccharides: galactose and N-acetylgalactosamine. The purified glycocalyx bound strongly to PNA lectin, fairly well to RCA120 lectin, and slightly to SBA lectin, but not to Con A, WGA, UEA-1, DBA, or LFA lectins. Exo-beta-galactosidase treatment increased SBA binding, whereas it decreased PNA binding. PNA was observed to strongly bind to the body surface of living EJ. The antigenic activity of the glycocalyx was remarkably lost by incubation with exo-beta-galactosidase and O-glycanase. The glycocalyx was reactive with sera of P. ohirai-infected rats, and its reactivity was remarkably reduced by O-glycanase treatment. The ELISA level was higher in sera at an early stage of infection than in a late one. These studies show that the EJ tegumental glycocalyx is antigenic in infection, a marked, high molecular weight glycoprotein containing antigenic O-linked sugars, and that the sugar epitope is at the nonreducing terminal of the O-linked sugars and is composed of galactose and N-acetylgalactosamine.

Animals↗
PubMed1985

The cercarial glycocalyx of Schistosoma mansoni.

Cercariae, the freshwater stage of Schistosoma mansoni infectious to man, are covered by a single unit membrane and an immunogenic glycocalyx. When cercariae penetrate the host skin, they transform to schistosomula by shedding tails, secreting mucous and enzymes, and forming microvilli over their surface. Here the loss of the glycocalyx from cercariae transforming in vitro was studied morphologically and biochemically. By scanning electron microscopy, the glycocalyx was a dense mesh composed of 15-30 nm fibrils that obscured spines on the cercarial surface. The glycocalyx was absent on organisms fixed without osmium and was partially lost when parasites aggregated in their own secretions before fixation. By transmission electron microscopy, a 1-2 microns thick mesh of 8-15-nm fibrils was seen on parasites incubated with anti-schistosomal antibodies or fixed in aldehydes containing tannic acid or ruthenium red. Cercariae transformed to schistosomula when tails were removed mechanically and parasites were incubated in saline. Within 5 min of transformation, organisms synchronously formed microvilli which elongated to 3-5 microns by 20 min and then were shed. However, considerable fibrillar material remained adherent to the double unit membrane surface of schistosomula. For biochemical labeling, parasites were treated with eserine sulfate, which blocked cercarial swimming, secretion, infectivity, and transformation to schistosomula. Material labeled by periodate oxidation and NaB3H4 was on the surface as shown by autoradiography and had an apparent molecular weight of greater than 10(6) by chromatography. Periodate-NaB3H4 glycocalyx had an isoelectric point of 5.0 +/- 0.4 and was precipitable with anti-schistosomal antibodies. More than 60% of the radiolabeled glycocalyx was released into the medium by transforming parasites in 3 h and was recovered as high molecular weight material. Parasites labeled with periodate and fluorescein-thiosemicarbazide and then transformed had a corona of fluorescence containing microvilli, much of which was shed onto the slide. Material on cercariae labeled by lodogen-catalyzed iodination was also of high molecular weight and was antigenic. In conclusion, the cercarial glycocalyx appears to be composed of acidic high molecular weight fibrils which are antigenic and incompletely cleared during transformation.

Animals↗