Genetic manipulation of blood group carbohydrates alters development and pathfinding of primary sensory axons of the olfactory systems

Primary sensory neurons in the vertebrate olfactory systems are characterised by the differential expression of distinct cell surface carbohydrates. We show here that the histo-blood group H carbohydrate is expressed by primary sensory neurons in both the main and accessory olfactory systems while the blood group A carbohydrate is expressed by a subset of vomeronasal neurons in the developing accessory olfactory system. We have used both loss-of-function and gain-of-function approaches to manipulate expression of these carbohydrates in the olfactory system. In null mutant mice lacking the alpha(1,2)fucosyltransferase FUT1, the absence of blood group H carbohydrate resulted in the delayed maturation of the glomerular layer of the main olfactory bulb. In addition, ubiquitous expression of blood group A on olfactory axons in gain-of-function transgenic mice caused mis-routing of axons in the glomerular layer of the main olfactory bulb and led to exuberant growth of vomeronasal axons in the accessory olfactory bulb. These results provide in vivo evidence for a role of specific cell surface carbohydrates during development of the olfactory nerve pathways. (c) 2006 Elsevier Inc. All rights reserved.

Topography of the combining region of a Thomsen-Friedenreich-antigen-specific lectin jacalin (Artocarpus integrifolia agglutinin). A thermodynamic and circular-dichroism spectroscopic study

Thermodynamic analysis of carbohydrate binding by Artocarpus integrifolia (jackfruit) agglutinin (jacalin) shows that, among monosaccharides, Me alpha GalNAc (methyl-alpha-N-acetylgalactosamine) is the strongest binding ligand. Despite its strong affinity for Me alpha GalNAc and Me alpha Gal, the lectin binds very poorly when Gal and GalNAc are in alpha-linkage with other sugars such as in A- and B-blood-group trisaccharides, Gal alpha 1-3Gal and Gal alpha 1-4Gal. These binding properties are explained by considering the thermodynamic parameters in conjunction with the minimum energy conformations of these sugars. It binds to Gal beta 1-3GalNAc alpha Me with 2800-fold stronger affinity over Gal beta 1-3GalNAc beta Me. It does not bind to asialo-GM1 (monosialoganglioside) oligosaccharide. Moreover, it binds to Gal beta 1-3GalNAc alpha Ser, the authentic T (Thomsen-Friedenreich)-antigen, with about 2.5-fold greater affinity as compared with Gal beta 1-3GalNAc. Asialoglycophorin A was found to be about 169,333 times stronger an inhibitor than Gal beta 1-3GalNAc. The present study thus reveals the exquisite specificity of A. integrifolia lectin for the T-antigen. Appreciable binding of disaccharides Glc beta 1-3GalNAc and GlcNAc beta 1-3Gal and the very poor binding of beta-linked disaccharides, which instead of Gal and GalNAc contain other sugars at the reducing end, underscore the important contribution made by Gal and GalNAc at the reducing end for recognition by the lectin. The ligand-structure-dependent alterations of the c.d. spectrum in the tertiary structural region of the protein allows the placement of various sugar units in the combining region of the lectin. These studies suggest that the primary subsite (subsite A) can accommodate only Gal or GalNAc or alpha-linked Gal or GalNAc, whereas the secondary subsite (subsite B) can associate either with GalNAc beta Me or Gal beta Me. Considering these factors a likely arrangement for various disaccharides in the binding site of the lectin is proposed. Its exquisite specificity for the authentic T-antigen, Gal beta 1-3GalNAc alpha Ser, together with its virtual non-binding to A- and B-blood-group antigens, Gal beta 1-3GalNAc beta Me and asialo-GM1 should make A. integrifolia lectin a valuable probe for monitoring the expression of T-antigen on cell surfaces.

Lewis phenotype, secretor status, and coeliac disease

Patients who cannot secrete ABO and Lewis blood group antigens into body fluids, an ability controlled by a single gene on chromosome 19, are known to be at increased risk of certain autoimmune diseases associated with human leucocyte antigen (HLA) markers. This study investigated the possibility of an association with coeliac disease using red cell Lewis (Le) blood group phenotype to infer secretor status. Among 73 patients with coeliac disease who had Le a or b antigen, 48% were non-secretors (Le a + b-) compared with 27% of 137 blood donors (p = 0.004: odds ratio 2.49, 95% confidence intervals 1.37 to 4.51) and 26% of 62 medical and nursing staff controls (p = 0.014: odds ratio 2.65, 95% confidence intervals 1.27 to 5.50). Clinical characteristics did not differ between secretors and non-secretors with coeliac disease. Thus, the non-secretor state is significantly associated with coeliac disease, suggesting that genes on chromosome 19 may directly or indirectly participate in conferring susceptibility.

Secretor status and Helicobacter pylori infection are independent risk factors for gastroduodenal disease

The hypothesis that non-secretors of ABO blood group antigens, a group shown to be more susceptible to certain bacterial infections, may be at greater risk of gastroduodenal disease because of increased susceptibility to Helicobacter pylori infection was investigated. Of 101 patients with symptoms of dyspepsia who were undergoing endoscopy, 32% were non-secretors (determined from Lewis blood group phenotype), 36% had endoscopically visible gastroduodenal disease (antral gastritis, gastric ulcer, erosive duodenitis, duodenal ulcer or some combination), and 58% had H pylori detected in antral biopsy specimens. Non-secretors and patients with H pylori infection were significantly more likely to have gastroduodenal disease (p = 0.02 and p = 0.002 respectively). There was, however, no significant association between secretor status and H pylori infection, logistic regression analysis confirming that these were independently associated with gastroduodenal disease. Overall, the relative risk of gastroduodenal disease for non-secretors compared with secretors was 1.9 (95% confidence intervals 1.2, 3.2). Non-secretion of ABO blood group antigens is not related to H pylori infection but is independently and significantly associated with endoscopic gastroduodenal disease. The mechanism of this remains to be explained.

Flow cytometric assessment of canine erythrocytes and platelets for dog erythrocyte antigen 1.1

Background: In human medicine, transfusion of ABO-mismatched platelets has been associated with shortened platelet survival and refractoriness to platelet transfusion because of expression of certain blood group antigens on platelets. It remains unknown if canine platelets express dog erythrocyte antigens (DEAs). Objective: The aim of this study was to develop a flow cytometric assay for DEA 1.1 and determine whether DEA 1.1 is present on canine platelets.Methods: Blood was collected from 172 clinically healthy dogs. Platelets and erythrocytes from each dog were tested for DEA 1.1 by flow cytometry using anti-DEA 1.1 blood-typing sera. Erythrocytes from each dog were also assessed for DEA 1.1 using a standard tube-typing test (T1) and using a second tube method (T2), if the flow cytometric and T1 results differed.Results: Using flow cytometry, DEA 1.1 was detected on erythrocytes of all 110 dogs shown by T1 or T2 testing to be DEA 1.1-positive. Initial results of the T1 test had a diagnostic accuracy of 93% (160 correct/ 172 tests). The frequency of erythrocyte DEA 1.1 positivity in previously untyped dogs (n = 118) was 56%. DEA 1.1 expression was not detected on platelets from DEA 1.1-positive dogs.Conclusions: Flow cytometry was a reliable method for detection of DEA 1.1 on canine erythrocytes. The absence of DEA 1.1 on platelets from DEA 1.1-positive dogs suggests that their platelets do not express DEA 1.1 and will not induce production of anti-DEA 1.1 antibodies that might lead to platelet refractoriness or reactions to a subsequent transfusion of DEA 1.1positive erythrocytes.

Expressão dos antígenos ABH e Lewis na gastrite crônica e alterações pré- neoplásica da mucosa gástrica

RACIONAL: A aderência do Helicobacter pylori à mucosa gástrica humana é pré-requisito para sua colonização e o desenvolvimento da gastrite crônica. Os antígenos de grupos sangüíneos, presentes no muco gástrico, são descritos como prováveis receptores da bactéria neste epitélio. A expressão alterada destes antígenos está associada ao desenvolvimento do câncer gástrico. OBJETIVOS: Verificar a ocorrência do Helicobacter pylori e a distribuição da expressão dos antígenos ABH e Lewis correlacionada com as alterações histopatológicas de pacientes com gastrite crônica. PACIENTES E MÉTODOS: Analisaram-se 63 amostras de sangue, saliva e biopsias gástricas de pacientes com gastrite crônica através das técnicas dot-blot-ELISA, imunoperoxidase indireta e colorações do Gram modificado e hematoxilina-eosina. RESULTADOS: Não foram encontradas associações significativas entre a presença da bactéria e os fenótipos de grupos sangüíneos ABH, Lewis e Secretor. Na maioria dos pacientes, a expressão dos antígenos ABH e Lewis, estava restrita principalmente ao epitélio foveolar da mucosa gástrica, concordando com a expressão ao nível salivar. A expressão inapropriada desses antígenos ocorria sempre na infecção pelo Helicobacter pylori e/ou alterações pré-neoplásicas da mucosa gástrica. Em áreas com metaplasia intestinal foi observada a redução da reatividade para os antígenos H e Le^b, e principalmente o aumento de Le^ª. CONCLUSÃO: Alterações no padrão de glicosilação destes antígenos refletem diferentes estágios de diferenciação celular e são marcadores potenciais na avaliação diagnóstica e prognóstica das patologias gástricas.

Aloimunização de doadores de sangue como fonte de anti-soros e hemácias raras

Pós-graduação em Pesquisa e Desenvolvimento (Biotecnologia Médica) - FMB

Modulare Synthese von Sialyl-Lewis X-Mimetika als Selektin-Liganden

Zelladhäsions- und Zellerkennungsphänomene spielen eine entscheidende Rolle im biologischen Geschehen: So findet die Kommunikation zwischen Zellen zu einem großen Teil zwischen membranständigen Adhäsionsmolekülen und ebenfalls membranständigen Liganden auf Nachbarzellen statt. Zu diesen Adhäsionsmolekülen gehören die Selektine, die von fundamentaler Bedeutung für die Bekämpfung von Entzündungskrankheiten und damit für die Funktionsweise unseres Immunsystems sind. Tritt eine Entzündung in unserem Körper auf, so sind Selektine an Adhäsionsprozessen beteiligt, die zur Auswanderung von Leukozyten aus dem Blutstrom in das entzündete Gewebe führen. Diese auswandernden Zellen enthalten eine Vielzahl von Wirkstoffen, die Krankheitserreger bekämpfen, aber auch körpereigenes Gewebe angreifen können. Viele Krankheitsbilder, die mit akut oder chronisch entzündlichen Prozessen einhergehen, hängen mit einer Dysregulation der Selektine zusammen. In diesen Fällen werden übermäßig Selektine exprimiert und es kommt zu einer lokal überschießenden Akkumulation von Leukozyten, die zu Schädigungen von gesundem Gewebe führen kann. rnZu diesen Krankheiten gehören z.B. die rheumatoide Arthritis, die myocardialen Ischämie, Psoriasis sowie Asthma und Allergien. Auch bei der Abstoßung von Transplantaten und Tumormetastasierung wurde eine Beteiligung der Selektine nachgewiesen. Eine Strategie gegen diese unerwünschten Effekte ist die selektive Inhibierung der Selektine, welche aufgrund der bedeutenden Rolle der Selektine in der Genese zahlreicher Krankheiten, von großem pharmazeutischen Interesse ist. rnIn der vorliegenden Arbeit ist die Entwicklung chemischer Synthesen von Verbindungen beschrieben, welche selektininhibierende Eigenschaften aufweisen sollen. Solche sog. Mimetika, die wie die natürlichen Liganden mit den Selektinen wechselwirken, sind nicht nur potenzielle Medikamente, sondern können auch durch veränderte Affinität zur Aufklärung von selektinvermittelten Zelladhäsionsprozessen dienen.rn

Multiplex suspension array for human anti-carbohydrate antibody profiling

Glycan-binding antibodies form a significant subpopulation of both natural and acquired antibodies and play an important role in various immune processes. They are for example involved in innate immune responses, cancer, autoimmune diseases, and neurological disorders. In the present study, a microsphere-based flow-cytometric immunoassay (suspension array) was applied for multiplexed detection of glycan-binding antibodies in human serum. Several approaches for immobilization of glycoconjugates onto commercially available fluorescent microspheres were compared, and as the result, the design based on coupling of end-biotinylated glycopolymers has been selected. This method requires only minute amounts of glycans, similar to a printed glycan microarray. The resulting glyco-microspheres were used for detection of IgM and IgG antibodies directed against ABO blood group antigens. The possibility of multiplexing this assay was demonstrated with mixtures of microspheres modified with six different ABO related glycans. Multiplexed detection of anti-glycan IgM and IgG correlated well with singleplex assays (Pearson's correlation coefficient r = 0.95-0.99 for sera of different blood groups). The suspension array in singleplex format for A/B trisaccharide, H(di) and Le(x) microspheres corresponded well to the standard ELISA (r > 0.94). Therefore, the described method is promising for rapid, sensitive, and reproducible detection of anti-glycan antibodies in a multiplexed format.

Genetic engineering of carbohydrate biosynthetic pathways in transgenic mice demonstrates cell cycle-associated regulation of glycoconjugate production in small intestinal epithelial cells.

Proliferation, migration-associated differentiation, and cell death occur continuously and in a spatially well-organized fashion along the crypt-villus axis of the mouse small intestine, making it an attractive system for studying how these processes are regulated and interrelated. A pathway for producing glycoconjugates was engineered in adult FVB/N transgenic mice by expressing a human alpha 1,3/4-fucosyltransferase (alpha 1,3/4-FT; EC 2.4.1.65) along the length of this crypt-villus axis. The alpha 1,3/4-FT can use lacto-N-tetraose or lacto-neo-N-tetraose core chains to generate Lewis (Le) blood group antigens Le(a) or Le(x), respectively, and H type 1 or H type 2 core chains to produce Leb and Le(y). Single- and multilabel immunohistochemical studies revealed that expression of the alpha 1,3/4-FT results in production of Le(a) and Leb antigens in both undifferentiated proliferated crypt cells and in differentiated postmitotic villus-associated epithelial cells. In contrast, Le(x) antigens were restricted to crypt cells. Villus enterocytes can be induced to reenter the cell cycle by expression of simian virus 40 tumor antigen under the control of a promoter that only functions in differentiated members of this lineage. Bitransgenic animals, generated from a cross of FVB/N alpha 1,3/4-FT with FVB/N simian virus 40 tumor antigen mice, expand the range of Le(x) expression to include villus-associated enterocytes that have reentered the cell cycle. Thus, the fucosylations unveil a proliferation-dependent switch in oligosaccharide production, as defined by a monoclonal antibody specific for the Le(x) epitope. These findings show that genetic engineering of oligosaccharide biosynthetic pathways can be used to define markers for entry into, or progression through, the cell cycle and to identify changes in endogenous carbohydrate metabolism that occur when proliferative status is altered in a manner that is not deleterious to the system under study.

The expression of ABH and Lewis antigens in Brazilian semi-isolated Black communities

The expression of the ABH and Lewis blood groups was determined in blood and saliva samples from two semi-isolated Black communities of Northern Brazil: Cametá and Alcântara. The distributions of ABO blood group phenotypes and the ABH secretor status frequencies showed no significant differences between these populations. In contrast, there was a difference regarding the frequency of the red blood cell Le(a-b-) phenotypes, associated with erythrocyte/saliva discordance, as confirmed by the observation that individuals with Le(a-b-) red cells have the Lewis antigen in their saliva, resulting in a nongenuine Le(a-b-) phenotype, whose frequency was higher in Alcântara.

Improving the detection of donations within the RHD negative blood donor panel which weakly express the RHD antigen

This project improved the detection and classification of very weakly expressed RhD variants in the Australian blood donor panel and contributed to the knowledge of anti-D reactivity patterns of RHD alleles that are undescribed. As such, the management of donations possessing these RHD alleles can be improved upon and the overall safety of transfusion medicine pertaining to the Rh blood group system will be increased. Future projects at ARCBS will be able to utilise the procedures developed in this project, thereby decreasing throughput time. The specificity of current testing will be improved and the need for outsourced RHD testing diminished.

Quantifying Cell Binding Kinetics Mediated By Surface-Bound Blood Type B Antigen To Immobilized Antibodies

Cell adhesion is crucial to many biological processes, such as inflammatory responses, tumor metastasis and thrombosis formation. Recently a commercial surface plasmon resonance (SPR)-based BIAcore biosensor has been extended to determine cell binding mediated by surface-bound biomolecular interactions. How such cell binding is quantitatively governed by kinetic rates and regulating factors, however, has been poorly understood. Here we developed a novel assay to determine the binding kinetics of surface-bound biomolecular interactions using a commercial BIAcore 3000 biosensor. Human red blood cells (RBCs) presenting blood group B antigen and CM5 chip bearing immobilized anti-B monoclonal antibody (mAb) were used to obtain the time courses of response unit, or sensorgrams, when flowing RBCs over the chip surface. A cellular kinetic model was proposed to correlate the sensorgrams with kinetic rates. Impacts of regulating factors, such as cell concentration, flow duration and rate, antibody-presenting level, as well as pH value and osmotic pressure of suspending medium were tested systematically, which imparted the confidence that the approach can be applied to kinetic measurements of cell adhesion mediated by surface-bound biomolecular interactions. These results provided a new insight into quantifying cell binding using a commercial SPR-based BIAcore biosensor.

Serological studies on oil sardine (Sardinella longiceps) and mackerel (Rastrelliger kanagurta)

An antiserum was raised in a rabbit against 0 panel red cells of mackerel. The erythrocytes of oil sardine and mackerel were tested against human blood typing sera anti A and B and also the test serum of rabbit which revealed the presence of antigens A and B. In addition, an antigen common to both the fishes and human A, B and 0 panel red cells was noted but not identifiable. The blood group B did not manifest itself clearly either in oil sardine or mackerel. The blood groups A, AB and 0 indicated the existence of genetically different groups of oil sardine and mackerel. Isoagglutinin tests revealed the presence of a reciprocal relationship with antigens A and B in both these fishes.

Corneal Allograft Rejection:Risk Factors, Diagnosis, Prevention, and Treatment

Recent advances in corneal graft technology, including donor tissue retrieval, storage and surgical techniques, have greatly improved the clinical outcome of corneal grafts. Despite these advances, immune mediated corneal graft rejection remains the single most important cause of corneal graft failure. Several host factors have been identified as conferring a "high risk" status to the host. These include: more than two quadrant vascularisation, with associated lymphatics, which augment the afferent and efferent arc of the immune response; herpes simplex keratitis; uveitis; silicone oil keratopathy; previous failed (rejected) grafts; "hot eyes"; young recipient age; and multiple surgical procedures at the time of grafting. Large grafts, by virtue of being closer to the host limbus, with its complement of vessels and antigen-presenting Langerhans cells, also are more susceptible to rejection. The diagnosis of graft rejection is entirely clinical and in its early stages the clinical signs could be subtle. Graft rejection is largely mediated by the major histocompatibility antigens, minor antigens and perhaps blood group ABO antigens and some cornea-specific antigens. Just as rejection is mediated by active immune mediated events, the lack of rejection (tolerance) is also sustained by active immune regulatory mechanisms. The anterior chamber associated immune deviation (ACAID) and probably, conjunctiva associated lymphoid tissue (CALT) induced mucosal tolerance, besides others, play an important role. Although graft rejection can lead to graft failure, most rejections can be readily controlled if appropriate management is commenced at the proper time. Topical steroids are the mainstay of graft rejection management. In the high-risk situations however, systemic steroids, and other immunosuppressive drugs such as cyclosporin and tacrolimus (FK506) are of proven benefit, both for treatment and prevention of rejection.