After traumatic spinal cord injury (SCI), disruption and plasticity of the microvasculature within injured spinal tissue contribute to the pathological cascades associated with the evolution of both primary and secondary injury. Conversely, preserved vascular function most likely results in tissue sparing and subsequent functional recovery. It has been difficult to identify subclasses of damaged or regenerating blood vessels at the cellular level. Here, adult mice received a single intravenous injection of the Griffonia simplicifolia isolectin B4 (IB4) at 1-28 days following a moderate thoracic (T9) contusion. Vascular binding of IB4 was maximally observed 7 days following injury, a time associated with multiple pathologic aspects of the intrinsic adaptive angiogenesis, with numbers of IB4 vascular profiles decreasing by 21 days postinjury. Quantitative assessment of IB4 binding shows that it occurs within the evolving lesion epicenter, with affected vessels expressing a temporally specific dysfunctional tight junctional phenotype as assessed by occludin, claudin-5, and ZO-1 immunoreactivities. Taken together, these results demonstrate that intravascular lectin delivery following SCI is a useful approach not only for observing the functional status of neovascular formation but also for definitively identifying specific subpopulations of reactive spinal microvascular elements. J. Comp. Neurol. 507:1031-1052, 2008.(c) 2007 Wiley-Liss, Inc.

Chicken embryos are an excellent model system for studies related to vascular morphogenesis. Development in ovo allows manipulations otherwise difficult in mammals, and the use of chicken-quail chimeras offers an additional advantage to this experimental system. Furthermore, the chicken chorioallantoic membrane has been extensively used for in vivo assays of angiogenesis. Surprisingly, few markers are available for a comprehensive visualization of the vasculature. Here we report the use of lectins for identification of embryonic chicken blood vessels. Nine lectins were evaluated using intravascular perfusion and directly on sections. Our results indicate that Lens culinaris agglutinin, concanavalin A, and wheat germ agglutinin can be used effectively for visualization of vessels of early chicken embryos (E2.5-E4). At later developmental stages, Lens culinaris agglutinin is a better choice because it displays equal affinity for the endothelia of arteries, veins, and capillaries. The findings presented here expand our understanding of lectin specificity in the endothelium of avian species and provide information as to the use of these reagents to obtain comprehensive labeling of the embryonic and chorioallantoic membrane vasculature.

A blood group B-specific lectin from the mushroom Marasmius oreades (MOA) was investigated with respect to its molecular structure and carbohydrate binding properties. SDS-PAGE mass spectrometric analysis showed it to consist of an intact (H; 33 kDa) and truncated (L; 23 kDa) subunit in addition to a small polypeptide (P; 10 kDa). Isolation in the presence of EDTA produced only the H subunits, indicating that the latter two are formed by metalloprotease cleavage of the intact H subunit. Tryptic digestion of the H, L, and P polypeptide chains followed by mass spectral analysis supports this view. The lectin strongly precipitated blood group type B substance, was nonreactive with type A substance, and reacted weakly with type H substance. Carbohydrate binding studies reveal a high affinity for Galalpha1,3Gal (but not for the isomeric alpha1,2-, alpha1,4-, and alpha1,6-disaccharides); Galalpha1,3Galbeta1,4GlcNAc; and the type B branched trisaccharide. MOA also reacts strongly with murine laminin from the Engelbreth-Holm-Swarm sarcoma and bovine thyroglobulin, both of which contain multiple Galalpha1,3Galbeta1,4GlcNAc end groups. This linear B trisaccharide is a component of porcine tissues and organs, preventing their transplantation into humans. MOA also shares carbohydrate recognition of this trisaccharide with toxin A elaborated by Clostridium difficile.

Quantification of changes in gastrointestinal morphology and mucus gel has been difficult to study. In the present study, we investigated changes in rat intestine under total parenteral nutrition (TPN) using fluoresceinated lectin staining and image analysis. Wistar rats (n = 34) were divided into two groups: one group received TPN for 2 weeks, and a control group received standard rat chow and water ad libitum for the same period. A 1-cm segment of distal ileum was removed and cut into cross sections. Sections were stained with hematoxylin and eosin, and to stain the mucus, periodic acid-Schiff (PAS), alcian blue (AB), and fluoresceinated lectin, that is, FITC-labeled Ulex europaeus agglutinin I (FITC-UEA-I), were used. Light microscope images were stored in a personal computer and analyzed using image analysis. We measured perimeter length, mucosal thickness, villus area, villus surface area index, mucus stain-positive area, mucosal area ratio, and mucosal surface area ratio. Perimeter length, mucosal thickness, villus area, and villus surface area index in the TPN group were significantly less than those in the control group (P < 0.001 for each parameter). In all mucus stainings, the stain-positive area in the TPN group was significantly less than that in the control group. However, there were no significant differences in mucosal area or mucosal surface area ratios between the two groups. The FITC-UEA-I-positive area was significantly greater than the PAS- or and AB-positive area. There were significant positive correlations between the FITC-UEA-I-positive area and both the PAS-positive and AB-positive areas. TPN for 2 weeks promoted intestinal atrophy and decreased absolute quantity of mucus gel. We successfully introduced the FITC-UEA-I staining method to evaluate changes in mucus gel.

Transplantation of porcine embryonic brain cells, including dopaminergic neurons, from ventral mesencephalon (VM) is considered a potential treatment for patients with Parkinson's disease. In the present study, we characterized the distribution among VM cells of the major porcine endothelial xenoantigen, the Galalpha1,3Gal epitope, and evaluated the cytotoxic effect of anti-Galalpha1,3Gal antibody-depleted and nondepleted human AB serum on VM cells. Overall levels of Galalpha1,3Gal-epitope expression was very low on the VM cell population using Bandeiraea simplicifolia IB(4) lectin staining of resuspended VM cells in flow cytometric analyses or staining of SDS-PAGE-separated, solubilized VM cell membrane proteins in Western blot analyses. Lectin-histochemical staining of sections of pig embryonal VM regions with BSA IB(4) lectin showed staining restricted to endothelial cells and microglia. In the presence of complement, both nondepleted and anti-Galalpha1,3Gal antibody-depleted AB sera were shown to be cytotoxic to VM cells as assessed in microcytotoxicity- and flow cytometry-based cytotoxicity assays. Purified IgM and IgG were both cytotoxic in the presence of complement. Three major VM cell membrane antigens of approximately 210, 105, and 50 kDa were reactive with natural IgM antibodies present in pooled human AB sera. Thus, antibody-dependent cytotoxicity may contribute to pig to human brain cell xenorejection, necessitating donor tissue modifications prior to a more widespread utilization of neural tissue xenografting.

The activity of seven different types of biotinylated lectin were examined in normal and tumorous lacrimal gland tissue. In the normal lacrimal gland tissue, the glandular cells and the tubular epithelium were labeled by maclura pomifera agglutinin (MPA), soybean agglutinin (SBA), and bauhinia purpurea agglutinin (BPA). The myoepithelial cells were stained by griffonia simplicifolia agglutinin 1 (GS1). In the primary pleomorphic adenoma tissue, the epithelial components were labeled by MPA, ulex europaeus agglutinin, SBA, peanut agglutinin, and BPA. The mesenchymal components showed negative labeling. In contrast, the recurrent pleomorphic adenoma tissue showed a positive reaction in the mesenchymal components when GS1 and BPA were used. These results revealed differences in the glycoconjugate composition among normal and tumorous lacrimal gland tissues from patients with primary and recurrent pleomorphic adenomas.

Pig to human xenotransplantation is considered a possible solution to the prevailing chronic lack of human donor organs for allotransplantation. The Galalpha1,3Gal determinant is the major porcine xenogeneic epitope causing hyperacute rejection following human antibody binding and complement activation. In order to characterize the tissue distribution of Galalpha1,3Gal-containing and blood group-type glycosphingolipids in pig, acid and nonacid glycosphingolipids were isolated from the kidney, small intestine, spleen, salivary gland, liver, and heart of a single pig obtained from a semi-inbred strain homozygous at the SLA locus. Glycolipids were analyzed by thin-layer immunostaining using monoclonal antibodies, and following ceramide glycanase cleavage as permethylated oligosaccharides by gas chromatography, gas chromatography-mass spectrometry, and matrix-assisted laser desorption/ionization mass spectrometry. The kidney contained large amounts of Galalpha1,3Gal-containing penta- and hexasaccharides having carbohydrate sequences consistent with the Galalpha1,3nLc4and Galalpha1,3Lexstructures, respectively. The former structure was tentatively identified in all organs by GC/MS. The presence of extended Galalpha1,3Gal-terminated structures in the kidney and heart was suggested by antibody binding, and GC/MS indicated the presence of a Galalpha1,3nLc6structure in the heart. The kidney, spleen, and heart contained blood group H pentaglycosylceramides based on type 1 (H-5-1) and type 2 (H-5-2) chains, and H hexaglycosylceramides based on the type 4 chain (H-6-4). In the intestine H-5-1 and H-6-4 were expressed, in the salivary gland H-5-1 and H-5-2, whereas only the H-5-1 structure was identified in the liver. Blood group A structures were identified in the salivary gland and the heart by antibody binding and GC/MS, indicating an organ-specific expression of blood group AH antigens in the pig.

The preclinical evaluation of polymer biocompatibility is often performed using animal subcutaneous implant models. The choice of subcutaneous tissue as the implant site is due to a number of factors including simplicity of the surgery involved. Results from subcutaneous implants cannot necessarily be extrapolated to other tissues due to the differences in cellular composition of tissues. We have evaluated and compared the healing characteristics of expanded polytetrafluoroethylene (ePTFE) discs implanted in either subcutaneous tissue or epididymal fat pad tissue in rats. Following 3 and 5 weeks of implantation, the healing characteristics of discs were evaluated histologically with particular emphasis on tissue and polymer neovascularization. Implants placed in subcutaneous tissue exhibited limited formation of new microvascular elements within and directly in contact with the polymer, and the formation of an extensive fibrous capsule. In contrast, ePTFE implanted in the epididymal fat pads of rats exhibited extensive neovascularization of tissue surrounding the polymer, penetration of these microvascular cells into the graft interstices for distances < or = 100 microns and no morphological evidence of a fibrous capsule. The rat epididymal fat pad provides an alternative tissue for polymer healing evaluations. Due to the extensive presence of fat in subcutaneous tissue in humans, we suggest the fat pad model provides a more relevant preclinical evaluation of the healing characteristics of polymers used clinically in anatomic positions which contain significant amounts of fat.

BACKGROUND: Alterations in microcirculation are considered central to the pathogenesis of hyperacute xenogeneic rejection (HXR) of vascularized xenografts, but currently there exist no data describing these microhemodynamic alterations. METHODS: Rat livers were perfused in situ with either isogeneic rat blood or xenogeneic human blood. The microcirculation of these xenoperfused livers was investigated directly using intravital fluorescence microscopy, and compared with that of isogeneic hemoperfused livers. In addition, the impact of antibody depletion by immunoadsorption was investigated. RESULTS: Although a homogenous microcirculation was found during isogeneic liver perfusion (index of acinar perfusion 90.4%/sinusoidal perfusion rate 93.6%), xenoperfusion resulted in a rapid breakdown of the microcirculation (47.5%/67.1%, respectively). Perfusion deficits were found predominantly in the periportal areas. Immunoadsorption reduced the total amount of IgM and IgG by 75.2% and 96.2%, respectively, and caused a significantly improved liver perfusion (80.2%/84.4%) and liver function, as indicated by bile production. In contrast, the massive hepatic leukocyte and platelet accumulation observed during perfusion with untreated xenogeneic blood was not altered by antibody depletion. CONCLUSIONS: Thus, the combination of isolated rat liver perfusion and intravital fluorescence microscopy enables the observation and quantification of the early phase of HXR. This is an important step forward for sensitive characterization of the rejection process and will enable the mechanisms involved in HXR to be elucidated. Antibody depletion was shown to improve liver function and perfusion, but did not reconstitute liver viability to the level of the isogeneic perfusion. These findings highlight the need for additional therapeutic regimens in xenografting.

Lectins are polyvalent proteins of non-immune origin with exquisite carbohydrate binding specificity making them ideal for investigation of cell surface glycoprotein and glycolipid antigens. We examined the cell surface lectin binding phenotypes of 20 UM-SCC squamous cell carcinoma cell lines established from 17 patients with head and neck cancers using a panel of fluorescein-conjugated lectins and inhibition by the appropriate monosaccharide to confirm specificity of using a panel of fluorescein-conjugated lectins and inhibition by the appropriate monosaccharide to confirm specificity of binding. Conconavalin A (Con A) from Canavalia ensiformis and the peanut agglutinin (PNA) from Arachis hypogaea bound all SCC cell lines tested and wheat germ agglutinin (WGA) from Triticum vulgaris bound to 12 of 13 tumor cell lines. The blood group O specific lectin UEA 1 from Ulex europeus also bound to all cell lines regardless of the donor blood type. Lectins of Dolichos biflorus (DBA) and Griffonia simplicifolia (GS I-B4 or BSA I-B4) with binding specificity for glycoproteins associated with blood group A and B respectively, had reactivity that did not directly correlate with blood group antigen expression. In contrast to the other lectins in our panel which exhibited broad reactivity with SCC antigens, the BSA-II lectin from Griffonia simplicfolia,(GS II or BSA II) which has sugar binding specificity for terminal non-reducing GlcNAc, did not bind to any of the screened cell lines. Our results demonstrate a common pattern of lectin-defined carbohydrate expression on the cell surface of squamous cell carcinomas of head and neck that appears promising in defining the malignant cellular phenotype. Lectin binding profile may be useful in differentiating benign from malignant histopathology.

Three fluorescein-labeled lectins have been shown to exhibit specificity for the surface of cells in different layers of the epidermis in the newborn rat. An isolectin from seeds of Bandeiraea simplicifolia with specificity for alpha-D-galactosyl end groups labeled the basal and lower spinous cells; a lectin from Ulex europaeus exhibiting specificity for alpha-L-fucosyl units outlines the surface of spinous cells, and a second lectin from B. simplicifolia, with specificity for N-acetyl-D-glucosamine, labels the cornified cells. Appropriate blocking experiments have confirmed the specific nature of the binding.

Laminin, a glycoprotein component of basal laminae, is synthesized and secreted in culture by a human malignant cell line (JAR) derived from gestational choriocarcinoma. Biosynthetically labeled human laminin subunits A (Mr approximately 400,000) and B (Mr = 200,000 doublet) are glycoslyated with asparagine-linked high mannose oligosaccharides that are processed to complex oligosaccharides before the laminin molecule is externalized by the cell. The rate-limiting step in the processing of the asparagine-linked glycans of laminin is at the point of action of alpha-mannosidase I since the principal laminin forms that accumulate in JAR cells contain Man9GlcNAc2 and Man8GlcNAc2 oligosaccharide units. The combination of subunits to form the disulfide-linked laminin molecule (Mr approximately 950,000) occurs rapidly within the cell at a time when the subunits contain these high mannose oligosaccharides. The production of laminin is limited by the availability of the A subunit such that excess B subunit forms accumulate intracellularly as uncombined B and a disulfide-linked B dimer. Pulse-chase kinetic studies establish these B forms as intermediates in the assembly of the laminin molecule. The fully assembled laminin undergoes further oligosaccharide processing and translocation to the cell surface, but uncombined B and B dimer are neither processed nor secreted to any significant extent. Therefore, laminin subunit combination appears to be a prerequisite for intracellular translocation, processing, and secretion. The mature laminin that contains complex oligosaccharides does not accumulate intracellularly but is rapidly externalized upon completion, either secreted into the culture medium (25%) or associated with the cell surface (75%) as determined by susceptibility to degradation by trypsin. About one-third of the laminin molecules secreted or shed by JAR cells into the chase medium contain a smaller A subunit form that appears to have been modified by limited proteolytic cleavage. The putative proteolytic event is closely timed to the release of the laminin into the culture medium.

An isolectin (BS I-B4) derived from Bandeiraea simplicifolia seeds and specific for terminal alpha-D-galactopyranosyl groups was found to be cytotoxic to Swiss 3T3 mouse cells. After mutagenesis and selection with BS I-B4, a variant clonal cell line resistant to both this isolectin and the alpha-D- and beta-D-galactose-binding lectin abrin was isolated. The parental cell line showed homogeneous and noninteracting binding sites for BS I-B4, whereas the variant cells exhibited a curved plot with a reduced number of binding regions. Another lectin, BS II, which is derived from the same seeds by specific for terminal N-acetyl-D-glucosaminyl groups, was cytotoxic to the variant but not the parental cells. These results suggest a possible lesion in the biosynthesis of cell surface structures resulting in the exposure of subterminal N-acetyl-D-glucosaminyl moieties in the variant line.

Evidence based on the quantitative precipitin method and hapten inhibition technique demonstrates that concanavalin A may interact with internal 2-O-linked alpha-D-mannopyranosyl residues as may occur in glycoproteins and polysaccharides.

A case of pulmonary infection, presenting with fever and productive cough (pseudohaemoptysis) was diagnosed as having infection with Serratia marcescens on performing culture and sensitivity tests. The organism was confirmed upto species level using the standard biochemical tests.

The interaction of the plant protein concanavalin A with glycogen and amylopectin was studied. The protein concentration-dependency of the interaction and the effect on the interaction of various enzymic and chemical degradations of the polysaccharides were investigated by using a turbidimetric technique. The results indicate that, in addition to certain structural requirements, the molecular weight of the polysaccharides and the concentration of protein are important factors controlling the extent of interaction. The observed similarity of the interaction of polysaccharides with concanavalin A to the more specific polysaccharide-protein interactions indicates that it should provide a useful model system for the study of these interactions in general.