Analysis by Surface Plasmon Resonance of the Influence of Valence on the Ligand Binding Affinity and Kinetics of an Anti-carbohydrate Antibody

The kinetics of ligand binding by Se155-4, an antibody specific for the Salmonella serogroup B O-polysaccharide, were studied by surface plasmon resonance. Because trace amounts of oligomers in Fab and single-chain antibody variable domain (scFv) preparations resulted in biphasic binding profiles that were difficult to analyze, all kinetic measurements were performed on purified monomeric fragments and, for certain mutant scFv, dimeric forms. Results obtained with monomeric forms indicated that the relatively low affinity of the antibody was due to rapid dissociation (koff approximately 0.25 s-1). Dimeric forms generally showed off-rates that were approximately 20-fold slower and a 5-fold increase in association rate constants to approximately 2 x 10(5) M-1 s-1. Although the association phases for scFv dimers showed good curve fitting to a one component interaction model, the dissociation phases were biphasic, presumably because the availability and accessibility of sites on the antigen always leads to some monovalent attachment. The fast off-rate for dimers was the same as the monomer off-rate. Se155-4 IgG off-rates were very similar to those observed for scFv dimer, whereas the onrate was the same as that obtained with Fab and scFv monomer

Selection of antibody single-chain variable fragments with improved carbohydrate binding by phage display.

A single-chain variable fragment (Fv) version of a murine monoclonal antibody, Se155-4, specific for Salmonella serogroup B O-polysaccharide, was used as a model system for testing monovalent phage display as a route for enhancing the relatively low affinities that typify anti-carbohydrate antibodies. Random single-chain Fv mutant libraries generated by chemical and error-prone polymerase chain reaction methods were panned against the serogroup B lipopolysaccharide. Panning of a randomly mutated heavy chain variable domain library indicated selection for improved serogroup B binders and yielded six mutants, five of which showed wild type activity by enzyme immunoassay. Two of these were apparently selected on the basis of better functional single-chain Fv yield in Escherichia coli. A heavy chain mutation (Ile77-->Thr) in one mutant, 3B1, appeared to have a particularly dramatic effect, resulting in yields of approximately 120 mg/liter of functional periplasmic product. The sixth mutant, 4B2, had complementarity determining region 1 (CDR1) and CDR2 mutations and demonstrated 10-fold improved binding, by enzyme immunoassay, relative to the wild type. Extensive analysis of antigen-antibody interactions indicated that the improved binding properties of 4B2 were attributable to a higher association rate constant and interaction with an epitope that is larger than the trisaccharide recognized by the wild type. None of the mutations involved known trisaccharide contact residues; this was consistent with analysis of wild type and mutant single-chain Fvs by titration microcalorimetry. Examination of the structure indicated that two mutations in the heavy chain CDR2 provided improved surface complementarity between the protein and the extended epitope encompassing 2 additional hexose residues. However, introduction of only the CDR2 mutations into the wild type structure failed to confer the improved binding properties of 4B2, indicating an indirect effect by the more distant mutations. Panning of randomly mutated light chain variable domain and full-length single-chain Fv mutant libraries did not yield mutants with improved assembly or binding properties

Bacterial expression and secretion of various single-chain Fv genes encoding proteins specific for a Salmonella serotype B O-antigen.

Active single-chain Fv molecules encoded by synthetic genes have been expressed and secreted to the periplasm of Escherichia coli using the ompA secretory signal. Four different constructs were developed to investigate the effects of peptide linker design and VL-VH orientation on expression, secretion, and binding to a Salmonella O-polysaccharide antigen. Peptide linker sequences derived from the elbow regions of the Fab molecule were used alone or in combination with the flexible (GGGGS)2 sequence. VL and VH domain order in the single chain molecules had a profound effect on the level of secretion but hardly influenced total expression levels, which were approximately 50 mg/liter, chiefly in the form of inclusion bodies. With VL in the NH2-terminal position, the amount of secreted product obtained was 2.4 mg/liter, but when VH occupied this position the yield was less than 5% of this value. Enzyme immunoassays of the four products showed domain order and linker sequence affected antigen binding by less than an order of magnitude. Attempts to express active Fv from dicistronic DNA were unsuccessful, but active Fv was obtained from single-chain Fv by enzymic cleavage at a site in the elbow linker peptide. The thermodynamic binding parameters of intact and cleaved single-chain Fvs determined by titration microcalorimetry were similar to those of bacterially produced Fab and mouse IgG

Supporting Aboriginal and Torres Strait Islander Families to Stay Together from the Start (SAFeST Start): Urgent call to action to address crisis in infant removals

Reducing the rate of over-representation of Aboriginal and Torres Strait Islander children in out-of-home care (OOHC) is a key Closing the Gap target committed to by all Australian governments. Current strategies are failing. The “gap” is widening, with the rate of Aboriginal and Torres Strait Islander children in OOHC at 30 June 2020 being 11 times that of non-Indigenous children. Approximately, one in five Aboriginal and Torres Strait Islander children entering OOHC each year are younger than one year. These figures represent compounding intergenerational trauma and institutional harm to Aboriginal and Torres Strait Islander families and communities. This article outlines systemic failures to address the needs of Aboriginal and Torres Strait Islander parents during pregnancy and following birth, causing cumulative harm and trauma to families, communities and cultures. Major reform to child and family notification and service systems, and significant investment to address this crisis, is urgently needed. The Family Matters Building Blocks and five elements of the Aboriginal and Torres Strait Islander Child Placement Principle (Prevention, Participation, Partnerships, Placement and Connection) provide a transformative foundation to address historical, institutional, well-being and socioeconomic drivers of current catastrophic trajectories. The time for action is now

Brucella abortus Induces the Premature Death of Human Neutrophils through the Action of Its Lipopolysaccharide

Most bacterial infections induce the activation of polymorphonuclear neutrophils (PMNs), enhance their microbicidal function, and promote the survival of these leukocytes for protracted periods of time. Brucella abortus is a stealthy pathogen that evades innate immunity, barely activates PMNs, and resists the killing mechanisms of these phagocytes. Intriguing clinical signs observed during brucellosis are the low numbers of Brucella infected PMNs in the target organs and neutropenia in a proportion of the patients; features that deserve further attention. Here we demonstrate that B. abortus prematurely kills human PMNs in a dose-dependent and cell-specific manner. Death of PMNs is concomitant with the intracellular Brucella lipopolysaccharide (Br-LPS) release within vacuoles. This molecule and its lipid A reproduce the premature cell death of PMNs, a phenomenon associated to the low production of proinflammatory cytokines. Blocking of CD14 but not TLR4 prevents the Br-LPS-induced cell death. The PMNs cell death departs from necrosis, NETosis and classical apoptosis. The mechanism of PMN cell death is linked to the activation of NADPH-oxidase and a modest but steadily increase of ROS mediators. These effectors generate DNA damage, recruitments of check point kinase 1, caspases 5 and to minor extent of caspase 4, RIP1 and Ca++ release. The production of IL-1β by PMNs was barely stimulated by B. abortus infection or Br-LPS treatment. Likewise, inhibition of caspase 1 did not hamper the Br-LPS induced PMN cell death, suggesting that the inflammasome pathway was not involved. Although activation of caspases 8 and 9 was observed, they did not seem to participate in the initial triggering mechanisms, since inhibition of these caspases scarcely blocked PMN cell death. These findings suggest a mechanism for neutropenia in chronic brucellosis and reveal a novel Brucella-host cross-talk through which B. abortus is able to hinder the innate function of PMN.Fondo Especial de la Educación Superior/[0500-13]/FEES/Costa RicaFondo Especial de la Educación Superior/[0504-13]/FEES/Costa RicaFondo Especial de la Educación Superior/[0505-13]/FEES/Costa RicaFondo Especial de la Educación Superior/[0248-13]/FEES/Costa RicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Centro de Investigación en Enfermedades Tropicales (CIET)UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)UCR::Vicerrectoría de Docencia::Salud::Facultad de Microbiologí