Benralizumab: eficacia y seguridad en pacientes con asma grave eosinofílica.

ResumenEl asma grave conlleva una carga de salud desproporcionadamente alta y cerca de la mitad de los adultos con esta patología tiene un fenotipo eosinofílico. En estos pacientes aunado a la producción de eosinófilos en médula ósea, se activan mecanismos de eosinopoyesis local en tejido pulmonar. Benralizumab es un anticuerpo monoclonal humanizado, que se une con alta afinidad y especificidad a la subunidad alfa del receptor de IL-5 (IL-5Rα) sobre la superficie de eosinófilos y otras células. El principal diferenciador de su mecanismo de acción se relaciona con la remoción de un residuo de fucosa en la Fc, lo cual incrementa hasta 50 veces la afinidad a células NK con apoptosis de eosinófilos mediante citotoxicidad celular dependiente de anticuerpos (CCDA), resultando en una reducción rápida y cercana al 100% tanto en suero como en médula ósea. Adicionalmente, benralizumab reduce >90% de los eosinófilos en tejido pulmonar y esputo. En diversos estudios clínicos controlados y en vida real se ha demostrado que esto se traduce en incremento actual del control del asma y disminución del riesgo futuro. El perfil de seguridad es adecuado sin haberse documentado infestaciones parasitarias ni efectos adversos a largo plazo relacionados con la reducción de los eosinófilos. Abstract Severe asthma carries a disproportionately high health burden and about half of adults with this pathology have an eosinophilic phenotype. In these patients, in addition to the production of eosinophils in bone marrow, local eosinopoiesis mechanisms are activated in lung tissue. Benralizumab is a humanized monoclonal antibody, which joins with high affinity and specificity to the alpha subunit of the IL-5 receptor (IL-5Rα) on the surface of eosinophils and other cells. The main differentiator of its mechanism of action is related to the removal of a fucose residue in Fc, which increases up to 50 times the affinity to NK cells with eosinophil apoptosis by antibody-dependent cell cytotoxicity (CCDA), that leads to a direct, rapid and nearly complete depletion in both peripheral blood and bone marrow. Additionally, benralizumab reduces >90% of eosinophils in lung tissue and sputum. Several controlled and real-life clinical studies have shown that this action over eosinophils is related to increased asthma control and decreased future risk. The safety profile is adequate without documenting parasitic infestations or long-term adverse effects related to the reduction of eosinophils.

Synthesis of Fucose Derivatives with Thiol Motifs towards Suicide Inhibition of Helicobacter pylori

The syntheses of six thiol-exhibiting monosaccharides towards suicide inhibition of Helicobacter pylori are reported. Blood group Antigen Binding Adhesin (BabA), a bacterial membrane-bound lectin, binds to human ABO and Lewis b blood group structures displayed on the surface of host epithelial cells. Crystal structures of the carbohydrate-recognition domain revealed a conserved disulfide bonded loop that anchors a critical fucose residue in these blood group structures. Disruption of this loop by N-acetylcysteine results in reduced BabA-mediated adherence to human gastric tissue sections and attenuated virulence in Lewis b-expressing transgenic mice. With a view of creating specific inhibitors of the lectin, we designed and successfully synthesised six fucose-derived compounds with thiol motifs to engage in a thiol-disulfide exchange with this disulfide bond of BabA and form a glycan-lectin disulfide linkage. Branching and extending the fucose backbone with 2- and 3-carbon thiol motifs delivered a range of candidates to be tested for biological activity against BabA.

Novel Rhamnosyltransferase Involved in Biosynthesis of Serovar 4-Specific Glycopeptidolipid from Mycobacterium avium Complex

ABSTRACT Glycopeptidolipids (GPLs) are one of the major glycolipid components present on the surface of Mycobacterium avium complex (MAC) that belong to opportunistic pathogens distributed in the natural environment. The serovars of MAC, up to around 30 types, are defined by the variable oligosaccharide portions of the GPLs. Epidemiological studies show that serovar 4 is the most prevalent type, and the prognosis of pulmonary disease caused by serovar 4 is significantly worse than that caused by other serovars. However, little is known about the biosynthesis of serovar 4-specific GPL, particularly the formation of the oligosaccharide portion that determines the properties of serovar 4. To investigate the biosynthesis of serovar 4-specific GPL, we focused on one segment that included functionally unknown genes in the GPL biosynthetic gene cluster of a serovar 4 strain. In this segment, a putative hemolytic protein gene, hlpA, and its downstream gene were found to be responsible for the formation of the 4-O-methyl-rhamnose residue, which is unique to serovar 4-specific GPL. Moreover, functional characterization of the hlpA gene revealed that it encodes a rhamnosyltransferase that transfers a rhamnose residue via 1→4 linkage to a fucose residue of serovar 2-specific GPL, which is a key pathway leading to the synthesis of oligosaccharide of serovar 4-specific GPL. These findings may provide clues to understanding the biological role of serovar 4-specific GPL in MAC pathogenicity and may also provide new insights into glycosyltransferase, which generates structural and functional diversity of GPLs.

Characterization of the Fucosylation Pathway in the Biosynthesis of Glycopeptidolipids from Mycobacterium avium Complex

ABSTRACT The cell envelopes of several species of nontuberculous mycobacteria, including the Mycobacterium avium complex, contain glycopeptidolipids (GPLs) as major glycolipid components. GPLs are highly antigenic surface molecules, and their variant oligosaccharides define each serotype of the M. avium complex. In the oligosaccharide portion of GPLs, the fucose residue is one of the major sugar moieties, but its biosynthesis remains unclear. To elucidate it, we focused on the 5.0-kb chromosomal region of the M. avium complex that includes five genes, two of which showed high levels of similarity to the genes involved in fucose synthesis. For the characterization of this region by deletion and expression analyses, we constructed a recombinant Mycobacterium smegmatis strain that possesses the rtfA gene of the M. avium complex to produce serovar 1 GPL. The results revealed that the 5.0-kb chromosomal region is responsible for the addition of the fucose residue to serovar 1 GPL and that the three genes mdhtA, merA, and gtfD are indispensable for the fucosylation. Functional characterization revealed that the gtfD gene encodes a glycosyltransferase that transfers a fucose residue via 1→3 linkage to a rhamnose residue of serovar 1 GPL. The other two genes, mdhtA and merA, contributed to the formation of the fucose residue and were predicted to encode the enzymes responsible for the synthesis of fucose from mannose based on their deduced amino acid sequences. These results indicate that the fucosylation pathway in GPL biosynthesis is controlled by a combination of the mdhtA, merA, and gtfD genes. Our findings may contribute to the clarification of the complex glycosylation pathways involved in forming the oligosaccharide portion of GPLs from the M. avium complex, which are structurally distinct.

Fuc(α1→3)GalNAc-: the major antigenic motif of Schistosoma mansoni glycolipids implicated in infection sera and keyhole-limpet haemocyanin cross-reactivity

The aim of the present study was the characterization of the dominant epitope present on Schistosoma mansoni glycolipids, which causes cross-reactivity of S. mansoni and S. haematobium infection sera with keyhole-limpet haemocyanin (KLH). To this end, the monoclonal antibody M2D3H was chosen for its similar behaviour in high-performance TLC immunostaining and inhibition-ELISA to infection sera. Individual, structurally defined oligosaccharides derived from S. mansoni egg glycolipids were tested for their binding to this monoclonal antibody by immunoaffinity chromatography. A terminal fucose residue linked in the (α1→3) position to N-acetylgalactosamine was found to be the common structural determinant of the four oligosaccharides binding to M2D3H. The Fuc(α1→3)GalNAc-motif also appeared to be the basis for the cross-reactivity with KLH, a phenomenon used in the serodiagnosis of S. mansoni, S. haematobium and S. japonicum infections.

Stochastic conformational search on the Lewis X (Lex) trisaccharide and three Lex analogues

Biased stochastic conformational searches using the MMFF94 force field and the Born continuum solvation model were applied to the molecular modeling of the Lewis X (Lex) trisaccharide (β-D-Gal-(1,4)-[α-L-Fuc-(1,3)]-β-D-GlcNAc-OH) and three Lex analogues, in which each of the three sugar units was replaced by another sugar residue, i.e., N-acetyl-glucosamine by glucose, galactose by glucose, and fucose by rhamnose. The stochastic search accurately identified a lowest energy conformation of the Lex determinant that corresponds to the reported conformations of Lex deduced experimentally in the solid state by X-ray crystallography and in solution by NMR measurements. In this conformation stacking exists between the galactosyl and fucosyl residues. Five new local minima for the Lex trisaccharide were found within 3 kcal mol–1 of the global minimum using the stochastic search and metric scaling. Modeling studies of the analogues showed that the stacking observed in the Lex trisaccharide was maintained when either galactosyl or N-acetylglucosamine were replaced by glucosyl residues. In contrast, substitution of the fucose residue by rhamnose led to two conformers in which stacking of the galactose and rhamnose residues was no longer maintained. These results indicate that the substitution of the non-reducing end galactosyl or N-acetyl-glucosaminyl residues by a glucose unit in the dimeric Lewis X (dimLex) tumour associated antigen could help in the development of a vaccine that cross-reacts with dimLex but no longer displays Lex associated three-dimensional epitopes also presented by non-cancerous cells. In contrast, an analogue in which the fucosyl residue is replaced by rhamnose does not constitute a good vaccine candidate, since our results indicate that this substitution will induce an important conformational change that is likely to abolish cross-reactivity with the natural dimLex tumor associated antigen.Key words: molecular modeling, stochastic, conformational analysis, Lewis X, oligosaccharide.

NolL of Rhizobium sp. Strain NGR234 Is Required for O-Acetyltransferase Activity

ABSTRACT Following (iso)flavonoid induction, nodulation genes of the symbiotic nitrogen-fixing bacterium Rhizobium sp. strain NGR234 elaborate a large family of lipooligosaccharidic Nod factors (NodNGR factors). When secreted into the rhizosphere of compatible legumes, these signal molecules initiate root hair deformation and nodule development. The nonreducing glucosamine residue of NodNGR factors are N acylated, N methylated, and mono- or biscarbamoylated, while position C-6 of the reducing extremity is fucosylated. This fucose residue is normally 2-O methylated and either sulfated or acetylated. Here we present an analysis of all acetylated NodNGR factors, which clearly shows that the acetate group may occupy position C-3 or C-4 of the fucose moiety. Disruption of the flavonoid-inducible nolL gene, which is preceded by anod box, results in the synthesis of NodNGR factors that lack the 3-O- or 4-O-acetate groups. Interestingly, the nodulation capacity of the mutant NGRΩnolL is not impaired, whereas introduction of thenod box::nolL construct into the related strain Rhizobium fredii USDA257 extends the host range of this bacterium to Calopogonium caeruleum,Leucaena leucocephala, and Lotus halophilus. Nod factors produced by a USDA257(pnolL) transconjugant were also acetylated. The nodbox::nolL construct was also introduced into ANU265 (NGR234 cured of its symbiotic plasmid), along with extra copies of the nodD1 gene. When permeabilized, these cells possessed acetyltransferase activity, although crude extracts did not.