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.

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.

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.

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.

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.