COMPLEX TREATMENT OF AMYOTROPHIC LATERAL SCLEROSIS PATIENT.

Amyotrophic lateral sclerosis is a progressive and fatal degenerative neuromuscular disease with few if any treatment options and physical rehabilitation addressing specific deficits is the most frequent form of therapy. Patients also suffer from depression and increased anxiety. Our purpose was to assess the neurorehabilitation effectiveness in a patient with amyotrophic lateral sclerosis who underwent stem cell transplantation but refused physiotherapy due to depression. Disease progression was followed using the revised Amyotrophic Lateral Sclerosis Functional Rating Scale bimonthly for six months pre- and then post-stem cell transplantation. Psychological traits were assessed using six standardized tests. Quantitative electroencephalogram diagnostics was performed before the first and after the last neurofeedback session, and sessions were conducted on a 3-times-a-week basis. The physiotherapy protocol included proprioceptive neuromuscular facilitation, electrical modalities unit applied to the lumbar spine area, and breathing, relaxation and walking exercises, among others. Increased motivation and marked decrease in the pain level was associated with the patient's willingness to complete physiotherapy, which resulted in improvements in most neuromuscular deficits and in increased respiratory capacity. During the 12 post-rehabilitation months, progression of the disease decelerated, and a positive behavioral change was noted. The study suggested that neurofeedback could be used as a neurorehabilitation component of the personalized complex rehabilitation protocol in patients with amyotrophic lateral sclerosis.

Migration of human mesenchymal stem cells stimulated with pulsed electric field and the dynamics of the cell surface glycosylation.

BACKGROUND: The analysis of the stem cells' glycome dynamics at different stages of differentiation and migration makes possible the exploration of the cell surface glycans as markers of the stem cell functional status, and, in the future, compatibility between transplanted cell and host environment. OBJECTIVES: The objective of our study was to develop novel techniques of investigating cell motility and to assess whether the electric field of the therapeutic spinal cord stimulation system used in vivo contributes to the migration of human mesenchymal stem cells (hMSCs) in vitro. MATERIAL AND METHODS: We have investigated the electrotaxis of bone marrow-derived MSCs using pulsed electric field (PEF) in the range of 16-80 mV/mm and the frequency of 130 Hz and 240 Hz. The PEF-related dynamics of the cell surface glycosylation was evaluated using 6 plant lectins recognizing individual glycans. RESULTS: Pulsed electric field at physiological levels (10 mV/mm; 130 Hz) did not influence cellular motility in vitro, which may correspond to the maintenance of the transplanted cells at the lesion site in vivo. An increase of the PEF intensity and the frequency exceeding physiological levels resulted in an increase in the cellular migration rate in vitro. Pulsed electric field elevated above physiological intensity and frequency (40-80 mV/mm; 240 Hz), but not at physiological levels, resulted in changes of the cell surface glycosylation. CONCLUSIONS: We found the described approach convenient for investigations and for the in vitro modeling of the cellular systems intended for the regenerative cell transplantations in vivo. Probing cell surface glycomes may provide valuable biomarkers to assess the competence of transplanted cells.

Natural Antibodies Against Sialoglycans.

Natural antibodies, part of the innate immunity system, are produced at strictly regulated levels in normal sera without immunization and thus are part of the innate immune system. The best studied natural antibodies are those directed against blood group antigens A and B and xeno-antigens including glycolylneuraminic acid containing Hanganutziu-Deicher (HD) glycolipid. Abnormal levels of anti-glycan antibodies were found in a number of pathologies. In many cases pathological antibodies are known to bind gangliosides. The genesis of anti-glycan antibodies in healthy humans and the reasons for their changes in pathologies are poorly understood. With a growing interest in their diagnostic applications, it is important to determine the carbohydrate structures that are recognized by antibodies present in the circulation of healthy individuals. We tested a large number of healthy donors using a printed glycan array (PGA) in a microchip format. The PGA contained ~300 glycans, representing mostly normal mammalian structures of glycoproteins and glycolipids, and many of the structures presented are biologically relevant sialylated motifs. As revealed by PGA, the sera interacted with at least 70 normal human glycans. With only few exceptions, antibodies recognizing sialosides have not been identified. Moderate levels of antibodies and moderate variability were observed in the case of SiaT n and its glycolyl variant. Unexpectedly, we found minimal antibody titer directed against Neu5Gcα and the trisaccharide Neu5Gcα2-6Galß1-4GlcNAc, although this form of neuraminic acid does not occur naturally in humans. Antibodies recognizing sialosides in unnatural ß-configuration have been detected and confirmed Springer's paradigm that circulating antibodies represent a reaction against bacteria. Gram-negative bacteria contain LPS with ßKDN and/or ßKDO which are very close analogs of Neu5Ac that are found in ß-connected form. Antibodies against the biantennary N-glycan chain, (Neu5Acα2-6Galß1-4GlcNAcß1-2Manα)2-3,6-Manß1-4GlcNAcß1-4GlcNAc were never observed and similarly we never saw antibodies directed against the SiaLe(a)/SiaLe (x) motifs. Anti-sialoglycan antibodies can be masked with gangliosides: for example, we observe about a five times higher level of anti-GD3 in purified total IgG compared to the same concentration of total Ig in the composition of native serum. For several antibodies we observed anomalous binding in diluted sera, namely, the signals towards sialylated glycans were increased in the PGA if diluted sera were used.

Glycomics, extracellular matrix, and anti-glycan antibodies in exfoliation syndrome.

Exfoliation syndrome (XFS) is considered to be a disease of extracellular matrix. Here we review key experimental evidence of aberrations in structure, expression, and function of glycoproteins, complex carbohydrates, and glycosaminoglycans found in extracellular matrix components forming exfoliation material in patients presenting with XFS. We hypothesize that certain components of the accumulating exfoliation material can become immunogenic, and multiple natural antibodies or autoantibodies are generated. Anti-glycan antibodies (AGAs) can be captured on Printed Glycan Array. Our preliminary results show robust immunoprofiles of AGAs in sera of patients with XFS, and the significant presence of AGAs in aqueous humor of these patients. These findings offer insight into the dynamics of AGAs during the development of XFS that could lead to the identification of the AGA-based XFS immuno-signature.

Cross-platform comparison of glycan microarray formats.

Carbohydrates participate in almost every aspect of biology from protein sorting to modulating cell differentiation and cell-cell interactions. To date, the majority of data gathered on glycan expression has been obtained via analysis with either anti-glycan antibodies or lectins. A detailed understanding of the specificities of these reagents is critical to the analysis of carbohydrates in biological systems. Glycan microarrays are increasingly used to determine the binding specificity of glycan-binding proteins (GBPs). In this study, six different glycan microarray platforms with different modes of glycan presentation were compared using five well-known lectins; concanavalin A, Helix pomatia agglutinin, Maackia amurensis lectin I, Sambucus nigra agglutinin and wheat germ agglutinin. A new method (universal threshold) was developed to facilitate systematic comparisons across distinct array platforms. The strongest binders of each lectin were identified using the universal threshold across all platforms while identification of weaker binders was influenced by platform-specific factors including presentation of determinants, array composition and self-reported thresholding methods. This work compiles a rich dataset for comparative analysis of glycan array platforms and has important implications for the implementation of microarrays in the characterization of GBPs.

Plasma Anti-Glycan Antibody Profiles Associated with Nickel level in Urine.

Nickel (Ni) compounds are widely used in industrial and commercial products including household and cooking utensils, jewelry, dental appliances and implants. Occupational exposure to nickel is associated with an increased risk for lung and nasal cancers, is the most common cause of contact dermatitis and has an extensive effect on the immune system. The purpose of this study was two-fold: (i) to evaluate immune response to the occupational exposure to nickel measured by the presence of anti-glycan antibodies (AGA) using a new biomarker-discovery platform based on printed glycan arrays (PGA), and (ii) to evaluate and compile a sequence of bioinformatics and statistical methods which are specifically relevant to PGA-derived information and to identification of putative "Ni toxicity signature". The PGAs are similar to DNA microarrays, but contain deposits of various carbohydrates (glycans) instead of spotted DNAs. The study uses data derived from a set of 89 plasma specimens and their corresponding demographic information. The study population includes three subgroups: subjects directly exposed to Nickel that work in a refinery, subjects environmentally exposed to Nickel that live in a city where the refinery is located and subjects that live in a remote location. The paper describes the following sequence of nine data processing and analysis steps: (1) Analysis of inter-array reproducibility based on benchmark sera; (2) Analysis of intra-array reproducibility; (3) Screening of data - rejecting glycans which result in low intra-class correlation coefficient (ICC), high coefficient of variation and low fluorescent intensity; (4) Analysis of inter-slide bias and choice of data normalization technique; (5) Determination of discriminatory subsamples based on multiple bootstrap tests; (6) Determination of the optimal signature size (cardinality of selected feature set) based on multiple cross-validation tests; (7) Identification of the top discriminatory glycans and their individual performance based on nonparametric univariate feature selection; (8) Determination of multivariate performance of combined glycans; (9) Establishing the statistical significance of multivariate performance of combined glycan signature. The above analysis steps have delivered the following results: inter-array reproducibility ρ=0.920 ± 0.030; intra-array reproducibility ρ=0.929 ± 0.025; 249 out of 380 glycans passed the screening at ICC>80%, glycans in selected signature have ICC ≥ 88.7%; optimal signature size (after quantile normalization)=3; individual significance for the signature glycans p=0.00015 to 0.00164, individual AUC values 0.870 to 0.815; observed combined performance for three glycans AUC=0.966, p=0.005, CI=[0.757, 0947]; specifity=94.4%, sensitivity=88.9%; predictive (cross-validated) AUC value 0.836.

Fibulin-3 as a blood and effusion biomarker for pleural mesothelioma.

BACKGROUND: New biomarkers are needed to detect pleural mesothelioma at an earlier stage and to individualize treatment strategies. We investigated whether fibulin-3 in plasma and pleural effusions could meet sensitivity and specificity criteria for a robust biomarker. METHODS: We measured fibulin-3 levels in plasma (from 92 patients with mesothelioma, 136 asbestos-exposed persons without cancer, 93 patients with effusions not due to mesothelioma, and 43 healthy controls), effusions (from 74 patients with mesothelioma, 39 with benign effusions, and 54 with malignant effusions not due to mesothelioma), or both. A blinded validation was subsequently performed. Tumor tissue was examined for fibulin-3 by immunohistochemical analysis, and levels of fibulin-3 in plasma and effusions were measured with an enzyme-linked immunosorbent assay. RESULTS: Plasma fibulin-3 levels did not vary according to age, sex, duration of asbestos exposure, or degree of radiographic changes and were significantly higher in patients with pleural mesothelioma (105±7 ng per milliliter in the Detroit cohort and 113±8 ng per milliliter in the New York cohort) than in asbestos-exposed persons without mesothelioma (14±1 ng per milliliter and 24±1 ng per milliliter, respectively; P<0.001). Effusion fibulin-3 levels were significantly higher in patients with pleural mesothelioma (694±37 ng per milliliter in the Detroit cohort and 636±92 ng per milliliter in the New York cohort) than in patients with effusions not due to mesothelioma (212±25 and 151±23 ng per milliliter, respectively; P<0.001). Fibulin-3 preferentially stained tumor cells in 26 of 26 samples. In an overall comparison of patients with and those without mesothelioma, the receiver-operating-characteristic curve for plasma fibulin-3 levels had a sensitivity of 96.7% and a specificity of 95.5% at a cutoff value of 52.8 ng of fibulin-3 per milliliter. In a comparison of patients with early-stage mesothelioma with asbestos-exposed persons, the sensitivity was 100% and the specificity was 94.1% at a cutoff value of 46.0 ng of fibulin-3 per milliliter. Blinded validation revealed an area under the curve of 0.87 for plasma specimens from 96 asbestos-exposed persons as compared with 48 patients with mesothelioma. CONCLUSIONS: Plasma fibulin-3 levels can distinguish healthy persons with exposure to asbestos from patients with mesothelioma. In conjunction with effusion fibulin-3 levels, plasma fibulin-3 levels can further differentiate mesothelioma effusions from other malignant and benign effusions. (Funded by the Early Detection Research Network, National Institutes of Health, and others.).

Repertoire of human natural anti-glycan immunoglobulins. Do we have auto-antibodies?

BACKGROUND: Profiling of donor's antibodies using glycan arrays demonstrated presence of antibodies capable of binding to >100 mammalian glycans or their fragments. For example, relatively high binding to Galα1-4Galß1-4GlcNAc (P(1)), Galα1-4Galß1-4Glc (P(k)), Galß1-3GlcNAc (Le(c)), 4-O-SuGalß1-4GlcNAc, and GalNAcα1-3GalNAc (Fs) was found in all tested individuals. Affinity isolation using hapten-specific chromatography in combination with epitope mapping revealed their glycotopes. Notably, a significant part of the antibodies was capable of recognizing a fragment of larger glycans, for example, -Galß1-4Glc of glycolipids, or Fucα1-3GlcNAc motif of Le(X)/Le(Y) antigens. Their epitope specificity did not vary between different healthy individuals. Nominally, all the mentioned immunoglobulins could be classified as auto-antibodies. METHODS: In this work we re-evaluated results published earlier and analyzed new data to address the question why autologous antibodies found in healthy individuals do not cause severe auto-immune reactions. RESULTS: In all cases the presumably "auto" antibodies were found to bind short fragments "subtracted" from larger glycans whereas recognition of the same fragment in the context of the whole natural chain was completely abolished. Thus, in spite of numerous formally positive signals observed on the printed glycan array, we are yet unable to identify in blood serum of healthy individuals true auto-antibodies capable of binding carbohydrate chains in their naturally occurring form. GENERAL SIGNIFICANCE: The identified natural anti-glycan antibodies were found to be specific, high-titer and population conservative immunoglobulins - all of this suggesting as yet unknown biological role(s) of the studied proteins. This article is part of a Special Issue entitled Glycoproteomics.

Serum antiglycan antibody detection of nonmucinous ovarian cancers by using a printed glycan array.

Epithelial ovarian cancer has the highest mortality rate among gynecological cancers. Altered glycosylation is associated with oncogenic transformation producing tumor-associated carbohydrate antigens. We investigated the potential of natural occurring antiglycan antibodies in the diagnosis of ovarian cancer by using printed glycan array. Antiglycan antibodies bound to 203 chemically synthesized printed glycans were detected via biotin-streptavidin fluorescence system in serum of women with normal operative findings (healthy controls; n = 24) and nonmucinous borderline or ovarian cancer of various FIGO stages (n = 33). Data were validated measuring blood group associated di-, tri and tetrasaccharide antigens on known ABO blood groups. Antiglycan antibodies demonstrated high reproducibility (r(c) > 0.9). Cluster analysis identified repetitive patterns of specific core carbohydrate structures: 11 N-linked glycans, 3 O-linked glycans and 2 glycosphingolipids. Biomarker detection revealed 24 glycans including P(1) (Galα1-4Galß1-4GlcNAcß; p < 0.001) significantly discriminating between (low-) malignant tumors and healthy controls. Comparable sensitivity and specificity with tumor marker CA125 was achieved by a panel of multivariate selected and linear combined antiglycan antibody signals (79.2 and 84.8%, respectively). Our findings demonstrate the potential of glycan arrays in the development of a new generation of biomarkers for ovarian cancer.

Processing and analysis of serum antibody binding signals from Printed Glycan Arrays for diagnostic and prognostic applications.

Procedures for data preprocessing, quality control, data analysis, evaluation and visualization of the new high-throughput biomarker platform based on printed glycan arrays (PGA) are presented in this paper. PGAs are similar in concept to DNA arrays but contain deposits of various carbohydrate structures (glycans) instead of spotted DNAs. PGA biomarker discovery for the early detection, diagnosis and prognosis of human malignancies and viral diseases is based on the response of the immune system as measured by the level of binding of anti-glycan antibodies from human serum to the glycans on the array. Procedures related to PGA data processing are herein demonstrated in a pilot study of cases representing 50 sera from patients with malignant mesothelioma and a control sample of 65 sera from high risk subjects exposed to asbestos without symptoms of disease.

Comparison of printed glycan array, suspension array and ELISA in the detection of human anti-glycan antibodies.

Anti-glycan antibodies represent a vast and yet insufficiently investigated subpopulation of naturally occurring and adaptive antibodies in humans. Recently, a variety of glycan-based microarrays emerged, allowing high-throughput profiling of a large repertoire of antibodies. As there are no direct approaches for comparison and evaluation of multi-glycan assays we compared three glycan-based immunoassays, namely printed glycan array (PGA), fluorescent microsphere-based suspension array (SA) and ELISA for their efficacy and selectivity in profiling anti-glycan antibodies in a cohort of 48 patients with and without ovarian cancer. The ABO blood group glycan antigens were selected as well recognized ligands for sensitivity and specificity assessments. As another ligand we selected P(1), a member of the P blood group system recently identified by PGA as a potential ovarian cancer biomarker. All three glyco-immunoassays reflected the known ABO blood groups with high performance. In contrast, anti-P(1) antibody binding profiles displayed much lower concordance. Whilst anti-P(1) antibody levels between benign controls and ovarian cancer patients were significantly discriminated using PGA (p=0.004), we got only similar results using SA (p=0.03) but not for ELISA. Our findings demonstrate that whilst assays were largely positively correlated, each presents unique characteristic features and should be validated by an independent patient cohort rather than another array technique. The variety between methods presumably reflects the differences in glycan presentation and the antigen/antibody ratio, assay conditions and detection technique. This indicates that the glycan-antibody interaction of interest has to guide the assay selection.

Detection of integrin-linked kinase in the serum of patients with malignant pleural mesothelioma.

OBJECTIVE: Integrin-linked kinase, which is relevant to neoplastic transformation, is highly expressed in malignant pleural mesothelioma. Recently, detection of integrin-linked kinase in serum of patients with ovarian cancer has been reported. This study asks whether integrin-linked kinase can also be detected in serum of patients with malignant pleural mesothelioma and whether serum level has diagnostic or prognostic relevance for that disease. METHODS: A sandwich enzyme-linked immunosorbent assay was designed to detect integrin-linked kinase and applied to serum samples from 46 patients with malignant pleural mesothelioma, 98 patients with other malignant chest disease, and 23 patients with benign chest disease. Integrin-linked kinase serum concentration and clinical data were correlated statistically. RESULTS: Median serum integrin-linked kinase concentration was significantly higher in malignant pleural mesothelioma (8.89 ng/mL) than in other malignant chest disease (0.66 ng/mL) or benign chest disease (0.78 ng/mL, P < .001). There was no relevant correlation of serum integrin-linked kinase with cell lysis parameters (R(2) < 0.1). Serum integrin-linked kinase concentration greater than 2.48 ng/mL had diagnostic sensitivity of 80%, specificity of 95%, positive predictive value of 85.7%, negative predictive value of 92.7%, and overall accuracy of 91% for distinction between malignant pleural mesothelioma and other diseases. Serum integrin-linked kinase concentration in malignant pleural mesothelioma was independent of histologic subtype or asbestos exposure. There was no statistically significant impact of serum integrin-linked kinase concentration on prognosis. CONCLUSIONS: Integrin-linked kinase can be detected in serum of patients with malignant pleural mesothelioma and may be a diagnostic marker for the disease.

Anti-carbohydrate antibodies of normal sera: findings, surprises and challenges.

We have used microchip format glycan array to characterize the individual carbohydrate recognition patterns by antibodies (Ab) in sera of 106 healthy donors. The glycan library included blood group antigens and other most frequent terminal oligosaccharides and their cores of mammalian N- and O-linked glycoproteins and glycolipids, tumor-associated carbohydrate antigens, and common components of bacterial/pathogenic polysaccharides and lipopolysaccharides, totally 205 glycans. The serum Ab interacted with at least 50 normal human glyco-motifs. Apart from expected blood group-, xeno- (heterophil) and infection-related binding activities, we observed a number of new and unexpected features. The surprising, relatively high antibody binding was found to the blood group P(1) and P(k) trisaccharides and H(type 2) trisaccharide. Novel and very high binding activities have been observed towards Galbeta1-3GlcNAc (Le(C)) related glycans, especially 3'-O-Su-Le(C), and towards 4'-O-sulfated lactosamine. Relatively high and uniform Ab binding to GalNAcalpha1-3Gal disaccharide demonstrated absence of correlation with fucosylated blood group A GalNAcalpha1-3(Fucalpha1-2)Gal antigen-similarly to well known relationship between Galalpha1-3Gal and true, fucosylated blood group B Galalpha1-3(Fucalpha1-2)Gal antigen. The binding intensity to Galalpha1-3Galbeta1-4GlcNAc xenoantigen was shown to be rather modest. Absence or very low Ab binding was found against oligosialic acid, sialooligosaccharides except SiaT(n), type 2 backbone glycans such as Le(y), and biantennary N-chain as well as its truncated forms, i.e. without terminal Sia, SiaGal, and SiaGalGlcNAc motifs. We have also found that Ab are capable of recognizing the short inner core typical for glycolipids (-Galbeta1-4Glc) and glycoproteins (-GalNAcalpha) as a fragment of bigger glycans.

Endogenous galectin-1 enforces class I-restricted TCR functional fate decisions in thymocytes.

During thymocyte development, the T-cell receptor (TCR) can discriminate major histocompatibility complex (MHC)/peptide ligands over a narrow range of affinities and translate subtle differences into functional fate decisions. How small differences in TCR input are translated into absolute differences in functional output is unclear. We examined the effects of galectin-1 ablation in the context of class-I-restricted thymocyte development. Galectin-1 expression opposed TCR partial agonist-driven positive selection, but promoted TCR agonist-driven negative selection of conventional CD8(+) T cells. Galectin-1 expression also promoted TCR agonist-driven CD8alphaalpha intestinal intraepithelial lymphocytes (IEL) development. Recombinant galectin-1 enhanced TCR binding to agonist/MHC complexes and promoted a negative-selection-signaling signature, reflected in intensified rapid and transient extracellular signal-regulated kinase (ERK) activation. In contrast, galectin-1 expression antagonized ERK activity in thymocytes undergoing positive selection. We propose that galectin-1 aids in discriminating TCR-directed fate decisions by promoting TCR binding to agonist/MHC complexes and enforcing agonist-driven signals, while opposing partial-agonist signals. In this way, galectin-1 widens the distinction between TCR-directed functional fate cues.

Printed covalent glycan array for ligand profiling of diverse glycan binding proteins.

Here we describe a glycan microarray constructed by using standard robotic microarray printing technology to couple amine functionalized glycans to an amino-reactive glass slide. The array comprises 200 synthetic and natural glycan sequences representing major glycan structures of glycoproteins and glycolipids. The array has remarkable utility for profiling the specificity of a diverse range of glycan binding proteins, including C-type lectins, siglecs, galectins, anticarbohydrate antibodies, lectins from plants and microbes, and intact viruses.

Fluorescence polarization as an analytical tool to evaluate galectin-ligand interactions.

Galectins are a family of beta-galactose binding lectins associated with functions such as immunological and malignant events. To study the binding affinity of galectins for natural and artificial saccharides and glycoconjugates we have developed an assay using fluorescence polarization. A collection of fluorescein-conjugated saccharides was synthesized and used as probes with galectins-1 and -3 and the two carbohydrate recognition domains of galectin-4. Direct binding of a fixed probe amount with different amounts of each galectin defined specificity and selectivity and permitted selection of the optimal probe for inhibition studies. Then fixed amounts of galectin and selected probe were used to screen the inhibitory potency of a library of nonfluorescent compounds. As the assay is in solution and does not require separation of free and bound probe, it is simple and rapid and can easily be applied to different unlabeled galectins. As all interaction components are known, K(d) values for galectin-inhibitor interaction can be directly calculated without approximation other than the assumption of a simple one-site competition.

Galectin-4 in normal tissues and cancer.

Galectin-4 belongs to a subfamily of galectins composed of two carbohydrate recognition domains within the same peptide chain. The two domains have all the conserved galectin signature amino acids, but their overall sequences are only approximately 40% identical. Both domains bind lactose with a similar affinity as other galectins, but their respective preferences for other disaccharides, and larger saccharides, are distinctly different. Thus galectin-4 has a property of a natural cross-linker, but in a modified sense since each domain prefers a different subset of ligands. Similarly to other galectins, galectin-4 is synthesized as a cytosolic protein, but can be externalized. During development and in adult normal tissues, galectin-4 is expressed only in the alimentary tract, from the tongue to the large intestine. It is often found in relatively insoluble complexes, as a component of either adherens junctions or lipid rafts in the microvillus membrane, and it has been proposed to stabilize these structures. Strong expression of galectin-4 can be induced, however, in cancers from other tissues including breast and liver. Within a collection of human epithelial cancer cell lines, galectin-4 is overexpressed and soluble in those forming highly differentiated polarized monolayers, but absent in less differentiated ones. In cultured cells, intracellular galectin-4 may promote resistance to nutrient starvation, whereas--as an extracellular protein--it can mediate cell adhesion. Because of its distinct induction in breast and other cancers, it may be a valuable diagnostic marker and target for the development of inhibitory carbohydrate-based drugs.

Galectin-3 binds lactosaminylated lipooligosaccharides from Neisseria gonorrhoeae and is selectively expressed by mucosal epithelial cells that are infected.

Galectins are a family of beta-galactoside binding proteins that have been proposed as host receptors for bacteria because beta-galactoside carbohydrates are common in bacterial membrane glycolipid lipooligosaccharides (LOS) and lipopolysaccharides. We investigated the interaction of galectin-3 with gonococcal LOS that make lactosyl (Lc2 or Lac), paraglobosyl (nLc4; LNnT; lacto-N-neotetraose), gangliosyl (IV3GalNAcnLc4), and neolactohexaosyl (nLc6, lactonorhexaosyl) oligosaccharides. All but gangliosyl LOS terminate in beta-galactoside. Galectin-3 had the highest affinity for the nLc6 LOS, which is made by a strain that is highly infectious for the male urethra, but also bound nLc4 LOS and to a Lac LOS. The lacto-N-neotetraose tetrasaccharide was a more potent inhibitor of galectin-3 binding to LOS than either lactose or N-acetyllactosamine. The relative affinity of galectin-3 for gonococci mirrored its affinity for purified LOS. Western blot analysis revealed expression of galectin-3 by human endometrial adenocarcinoma and prostatic epithelial cells that can be invaded by gonococci. Immunohistochemistry of human fallopian tube epithelium showed localized expression of galectin-3 by non-ciliated cells, the specific cell gonococci invade in this tissue. We conclude that because of its location and affinity for gonococcal LOS galectin-3 could play a role in gonococcal infection.

Regulation of mucosal immune responses by recombinant interleukin 10 produced by intestinal epithelial cells in mice.

BACKGROUND & AIMS: Interleukin (IL)-10 is a cytokine with anti-inflammatory properties. The aim of this study was to explore the effect of a site-specific delivery of IL-10 on intestinal immune responses. METHODS: Transgenic mice were created in which IL-10 is expressed by the intestinal epithelial cells. RESULTS: Transgenic mice showed a marked increase in the number of intraepithelial lymphocytes in the small intestine. Mucosal lymphocytes of transgenic animals produced fewer T helper type 1 cytokines than wild-type lymphocytes. By contrast, the production of transforming growth factor beta was increased. Moreover, the epithelial layer in transgenic mice was significantly enriched for CD4(+)CD25(+) T cells. Furthermore, transgenic mice had increased numbers of immunoglobulin A-producing B cells in the small intestine. These effects were local because splenic lymphocytes were not affected. Studies in models of inflammatory bowel disease showed that transgenic IL-10 was able to attenuate the acute colitis induced by dextran sodium sulfate administration or by adoptive transfer of CD4(+)CD45RB(high) splenocytes, with a modest effect on the chronic intestinal inflammation arising spontaneously in IL-10(-/-) mice. CONCLUSIONS: These observations provide evidence for an in vivo lymphoepithelial cross talk, by which cytokines locally produced by epithelial cells can regulate immune responses in the intestine without systemic modifications.