Synthesis of neoglycoconjugates and oligosaccharides with potential anti-Helicobacter pylori activity

The significance of carbohydrate-protein interactions in many biological phenomena is now widely acknowledged and carbohydrate based pharmaceuticals are under intensive development. The interactions between monomeric carbohydrate ligands and their receptors are usually of low affinity. To overcome this limitation natural carbohydrate ligands are often organized as multivalent structures. Therefore, artificial carbohydrate pharmaceuticals should be constructed on the same concept, as multivalent carbohydrates or glycoclusters. Infections of specific host tissues by bacteria, viruses, and fungi are among the unfavorable disease processes for which suitably designed carbohydrate inhibitors represent worthy targets. The bacterium Helicobacter pylori colonizes more than half of all people worldwide, causing gastritis, gastric ulcer, and conferring a greater risk of stomach cancer. The present medication therapy for H. pylori includes the use of antibiotics, which is associated with increasing incidence of bacterial resistance to traditional antibiotics. Therefore, the need for an alternative treatment method is urgent. In this study, four novel synthesis procedures of multivalent glycoconjugates were created. Three different scaffolds representing linear (chondroitin oligomer), cyclic (γ-cyclodextrin), and globular (dendrimer) molecules were used. Multivalent conjugates were produced using the human milk type oligosaccharides LNDFH I (Lewis-b hexasaccharide), LNnT (Galβ1-4GlcNAcβ1-3Galβ1-4Glc), and GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc all representing analogues of the tissue binding epitopes for H. pylori. The first synthetic method included the reductive amination of scaffold molecules modified to express primary amine groups, and in the case of dendrimer direct amination to scaffold molecule presenting 64 primary amine groups. The second method described a direct procedure for amidation of glycosylamine modified oligosaccharides to scaffold molecules presenting carboxyl groups. The final two methods that were created both included an oxime-linkage on linkers of different length. All the new synthetic procedures synthesized had the advantage of using unmodified reducing sugars as starting material making it easy to synthesize glycoconjugates of different specificity. In addition, the binding activity of an array of neoglycolipids to H. pylori was studied. Consequently, two new neolacto-based structures, Glcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-Cer and GlcAβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-Cer, with binding activity toward H. pylori were discovered. Interestingly, N-methyl and N-ethyl amide modification of the GlcAβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-Cer glucuronic acid residue resulted in more effective H. pylori binding epitopes than the parent molecule.Yli puolet maailman väestöstä kantaa Gram-negatiivistä Helicobacter pylori bakteeria. Helikobakteeri-infektio hankitaan yleensä lapsuusiässä, tartunta saadaan ilmeisimmin vanhemmilta tai isovanhemmilta. Kehittyneissä maissa lasten helikobakteeri-infektio on harvinainen verrattuna sosioekonomisesti vähemmän kehittyneisiin maihin. Mahalaukun limakalvossa olevan helikobakteeri-infektion kaikille kantajille kehittyy krooninen gastriittinen tulehdus, mutta tämä tila on usein oireeton. Tulehdus johtaa noin 10-20%:lla potilaista vuosien kuluessa maha- tai pohjukaissuolihaavan kehittymiseen. Helikobakteeri-infektio lisää mahasyövän riskiä 2-6 kertaiseksi ja sitä pidetään keskeisenä tekijänä mahan MALT-lymfooman synnyssa. Infektion saaneista 10-20%:lle bakteerikolonisaation lopputulos voi olla hengenvaarallinen. Vuonna 1994 WHO (World Health Organization) kategorisoi helikobakteerin korkeimman luokan karsinogeeniksi. Vaikka helikobakteerin häätöhoitoon tällä hetkellä käytettävä hoito on varsin tehokas, ongelmina ovat antibiooteille resistenssien kantojen yleistyminen ja pahat sivuvaikutukset, kuten ruoansulatusjärjestelmän normaalin bakteeriflooran tuhoutuminen. Antibioottien käytöstä suuremmalle populaatiolle helikobakteerin häätöhoidossa seuraisi antibiooteille resistenssien kantojen yleistyminen entisestään. Helikobakteeri-infektion aiheuttama gastriitti, tästä edelleen aiheutuvat taudit ja syövän riskin kasvu ovat kuitenkin vakava terveysongelma, joten uusille terapiahoidoille on tarve. Helikobakteeri infektoi tarttumalla mahan pinnan hiilihydraattirakenteisiin. Ihmisen todennäköisesti kaksi merkittävintä helikobakteerin reseptorihiilihydraattia on nyt tunnistettu. Liukoista reseptorin analogia voidaan käyttää helikobakteerin irrottamiseen mahan pinnasta. Näitä monovalenttisia reseptorianalogeja tarvitaan epäkäytännöllisen korkea määrä estämään patogeenin tarttuminen ihmiseen, kun sensijaan luonnonmukaiseen sokerirunkoon rakennetut multivalentit molekyylit voivat olla jopa miljoona kertaa tehokkaampia. Tämän tutkimuksen tavoitteena on ollut kehittää menetelmiä polyvalenttisten oligosakkaridikonjugaattien valmistamiseksi. Menetelmien todentamiseksi oli myös valmistettava polyvalenttirungot, joihin oligosakkaridiyksiköt kiinnitetään. Lisäksi tavoitteena on ollut rakentaa potentiaalisia uusia monovalenttisia helikobakteerin reseptorianalogeja ja tutkia näiden sitoutumista helikobakteeriin in vitro. Tutkimuksessa on kehitetty neljä erilaista prosessia oligosakkaridien kiinnittämiseen hiilihydraattirunkoihin multivalenteiksi rakenteiksi. Runkorakenteita on kehitetty muokkaamalla kemiallisesti luonnollisia hiilihydraattirunkoja (γ-syklodekstriiniä ja kondroitiinisulfaattia). Kaikkien näihin runkoihin kiinnitettyjen hiilihydraattirakenteiden on aikaisemmin todettu tarttuvan helikobakteeriin. Helikobakteerin häätöhoidon lisäksi näitä multivalenttipohjia on mahdollista soveltaa muidenkin hiilihydraatteihin sitoutuvien patogeenien sitoutumisen estoon liittämällä näihin tapauskohtainen hiilihydraattiepitooppi. Lisäksi tutkittiin, in vitro, paneeli potentiaalisia uusia monovalenttisia helikobakteerin reseptorianalogeja, joista löydettiin kaksi uutta helikobakteeria sitovaa rakennetta

Expression and Regulation of Latent TGF-β Binding Protein-1 Transcripts and Their Splice Variants in Human Glomerular Endothelial Cells

Latent transforming growth factor (TGF)-β-binding protein (LTBP) is required for the assembly, secretion, matrix association, and activation of latent TGF-β complex. To elucidate the cell specific expression of the genes of LTBP-1 and their splice variants and the factors that regulate the gene expression, we cultured primary human glomerular endothelial cells (HGEC) under different conditions. Basal expression of LTBP-1 mRNA was suppressed in HGEC compared to WI-38 human embryonic lung fibroblasts. High glucose, H2O2, and TGF-β1 upregulated and vascular endothelial growth factor (VEGF) further downregulated LTBP-1 mRNA in HGEC. RT-PCR with a primer set for LTBP-1S produced many clones but no clone was gained with a primer set for LTBP-1L. Of 12 clones selected randomly, Sca I mapping and DNA sequencing revealed that only one was LTBP-1S and all the others were LTBP-1SΔ53. TGF-β1, but not high glucose, H2O2 or VEGF, tended to increase LTBP-1SΔ53 mRNA. In conclusion, HGEC express LTBP-1 mRNA which is suppressed at basal state but upregulated by high glucose, H2O2, and TGF-β1 and downregulated by VEGF. Major splice variant of LTBP-1 in HGEC was LTBP-1SΔ53. Modification of LTBP-1SΔ53 gene in HGEC may abrogate fibrotic action of TGF-β1 but this requires confirmation

Single-Step Grafting of Aminooxy-Peptides to Hyaluronan: A Simple Approach to Multifunctional Therapeutics for Experimental Autoimmune Encephalomyelitis

The immune response to antigens is directed in part by the presence or absence of costimulatory signals. The ability to coincidently present both antigen and, for example, a peptide that inhibits or activates the costimulatory pathway, would be a valuable tool for tolerization or immunization, respectively. A simple reaction scheme utilizing oxime chemistry was identified as a means to efficiently conjugate different peptide species to hyaluronan. Peptides synthesized with an aminooxy N-terminus reacted directly to hyaluronan under slightly acidic aqueous conditions without the need for a catalyst. The resulting oxime bond was found to rapidly hydrolyze at pH 2 releasing peptide, but was stable at higher pH values (5.5 and 7). Two different peptide species, a multiple sclerosis antigen (PLP) and an ICAM-1 ligand (LABL) known to block immune cell stimulation, were functionalized with the aminooxy end group. These peptides showed similar reactivity to hyaluronan and were conjugated in an equimolar ratio. The resulting hyaluronan with grafted PLP and LABL significantly inhibited disease in mice with experimental autoimmune encephalomyelitis, a model of multiple sclerosis. Aminooxy-peptides facilitate simple synthesis of multifunctional hyaluronan graft polymers, thus enabling novel approaches to antigen-specific immune modulation