Simon Chapman and Ross MacKenzie review the evidence and argue that health promotion messages should emphasize that the most successful method used by most ex-smokers is unassisted cessation.
The lipopolysaccharide (LPS)-binding protein (LBP) has a concentration-dependent dual role in the pathogenesis of gram-negative sepsis: low concentrations of LBP enhance the LPS-induced activation of mononuclear cells (MNC), whereas the acute-phase rise in LBP concentrations inhibits LPS-induced cellular stimulation. In stimulation experiments, we have found that LBP mediates the LPS-induced cytokine release from MNC even under serum-free conditions. In biophysical experiments we demonstrated that LBP binds and intercalates into lipid membranes, amplified by negative charges of the latter, and that intercalated LBP can mediate the CD14-independent intercalation of LPS into membranes in a lipid-specific and temperaturedependent manner. In contrast, prior complexation of LBP and LPS inhibited binding of these complexes to membranes due to different binding of LBP to LPS or phospholipids. This results in a neutralization of LPS and, therefore, to a reduced production of tumor necrosis factor by MNC. We propose that LBP is not only present as a soluble protein in the serum but may also be incorporated as a transmembrane protein in the cytoplasmic membrane of MNC and that the interaction of LPS with membrane-associated LBP may be an important step in LBP-mediated activation of MNC, whereas LBP-LPS complexation in the serum leads to a neutralization of LPS.Human lipopolysaccharide (LPS)-binding protein (LBP) is a serum glycoprotein belonging to a family of lipid-binding proteins which includes bactericidal/permeability-increasing protein (BPI), phospholipid ester transfer protein, and cholesterol ester transfer protein (1,18,36). It consists of 456 amino acid residues preceded by a hydrophobic signal sequence of 25 residues (31). LBP is synthesized by hepatocytes (26) and intestinal epithelial cells (42) and is present in normal serum at concentrations of 5 to 10 g/ml, rising up to 200 g/ml 24 h after induction of an acute-phase response (35). This rise in LBP levels is caused by transcriptional activation of the LBP gene mediated by interleukin-1 (IL-1) and . LBP has a concentration-dependent dual role: low concentrations of LBP enhance the LPS-induced activation of mononuclear cells (MNC), whereas the acute-phase rise in LBP concentrations inhibits LPS-induced cellular stimulation (20). LBP binds a variety of LPS (endotoxin) chemotypes from rough and smooth strains of gram-negative bacteria and even lipid A, the lipid moiety of LPS (37, 38). The LPS molecules, components of the outer membrane of gram-negative bacteria, are important mediators in the pathogenesis of gram-negative sepsis and septic shock (25). Because the lipid A moiety has been shown to be responsible for the biological activity of LPS in most in vivo and in vitro test systems, it has been termed the endotoxic principle of LPS (27).LPSs activate monocytes and macrophages to secrete inflammatory cytokines (tumor necrosis factor alpha [TNF-␣] and IL-1, etc.) and other potent mediators (32) by an intracellular signal amplification pathway. These mediato...
Clostridium difficile is a leading cause of nosocomial infection in North AmericaWith favorable characteristics such as high production yield, potent toxin neutralization, and intrinsic stability, these V H Hs are attractive systemic therapeutics but are more so as oral therapeutics in the destabilizing environment of the gastrointestinal tract.
SYNOPSISIn awake human subjects, neural responses in radial nerves to electrical stimulation were recorded with intrafascicular tungsten microelectrodes. Changes in the activity of individual fibre groups during blocking procedures were recorded and correlated with simultaneous alterations in the perception of standardized stimuli. Light touch sensibility in hairy skin appeared to depend on the integrity of A-beta-gamma fibres, cold and pinprick on A-delta fibres, and warmth and dull pain on C fibres.
The extreme pH and protease-rich environment of the upper gastrointestinal tract is a major obstacle facing orally-administered protein therapeutics, including antibodies. Through protein engineering, several Clostridium difficile toxin A-specific heavy chain antibody variable domains (VHHs) were expressed with an additional disulfide bond by introducing Ala/Gly54Cys and Ile78Cys mutations. Mutant antibodies were compared to their wild-type counterparts with respect to expression yield, non-aggregation status, affinity for toxin A, circular dichroism (CD) structural signatures, thermal stability, protease resistance, and toxin A-neutralizing capacity. The mutant VHHs were found to be well expressed, although with lower yields compared to wild-type counterparts, were non-aggregating monomers, retained low nM affinity for toxin A, albeit the majority showed somewhat reduced affinity compared to wild-type counterparts, and were capable of in vitro toxin A neutralization in cell-based assays. Far-UV and near-UV CD spectroscopy consistently showed shifts in peak intensity and selective peak minima for wild-type and mutant VHH pairs; however, the overall CD profile remained very similar. A significant increase in the thermal unfolding midpoint temperature was observed for all mutants at both neutral and acidic pH. Digestion of the VHHs with the major gastrointestinal proteases, at biologically relevant concentrations, revealed a significant increase in pepsin resistance for all mutants and an increase in chymotrypsin resistance for the majority of mutants. Mutant VHH trypsin resistance was similar to that of wild-type VHHs, although the trypsin resistance of one VHH mutant was significantly reduced. Therefore, the introduction of a second disulfide bond in the hydrophobic core not only increases VHH thermal stability at neutral pH, as previously shown, but also represents a generic strategy to increase VHH stability at low pH and impart protease resistance, with only minor perturbations in target binding affinities. These are all desirable characteristics for the design of protein-based oral therapeutics.
One of the major causes of morbidity and mortality in man and economically important animals is bacterial infections of the gastrointestinal (GI) tract. The emergence of difficult-to-treat infections, primarily caused by antibiotic resistant bacteria, demands for alternatives to antibiotic therapy. Currently, one of the emerging therapeutic alternatives is the use of lytic bacteriophages. In an effort to exploit the target specificity and therapeutic potential of bacteriophages, we examined the utility of bacteriophage tailspike proteins (Tsps). Among the best-characterized Tsps is that from the Podoviridae P22 bacteriophage, which recognizes the lipopolysaccharides of Salmonella enterica serovar Typhimurium. In this study, we utilized a truncated, functionally equivalent version of the P22 tailspike protein, P22sTsp, as a prototype to demonstrate the therapeutic potential of Tsps in the GI tract of chickens. Bacterial agglutination assays showed that P22sTsp was capable of agglutinating S. Typhimurium at levels similar to antibodies and incubating the Tsp with chicken GI fluids showed no proteolytic activity against the Tsp. Testing P22sTsp against the three major GI proteases showed that P22sTsp was resistant to trypsin and partially to chymotrypsin, but sensitive to pepsin. However, in formulated form for oral administration, P22sTsp was resistant to all three proteases. When administered orally to chickens, P22sTsp significantly reduced Salmonella colonization in the gut and its further penetration into internal organs. In in vitro assays, P22sTsp effectively retarded Salmonella motility, a factor implicated in bacterial colonization and invasion, suggesting that the in vivo decolonization ability of P22sTsp may, at least in part, be due to its ability to interfere with motility… Our findings show promise in terms of opening novel Tsp-based oral therapeutic approaches against bacterial infections in production animals and potentially in humans.
Small recombinant antibody fragments (e.g. scFvs and VHHs), which are highly tissue permeable, are being investigated for antivenom production as conventional antivenoms consisting of IgG or F(ab’)2 antibody fragments do not effectively neutralize venom toxins located in deep tissues. However, antivenoms composed entirely of small antibody fragments may have poor therapeutic efficacy due to their short serum half-lives. To increase serum persistence and maintain tissue penetration, we prepared low and high molecular mass antivenom antibodies. Four llama VHHs were isolated from an immune VHH-displayed phage library and were shown to have high affinity, in the low nM range, for α-cobratoxin (α–Cbtx), the most lethal component of Naja kaouthia venom. Subsequently, our highest affinity VHH (C2) was fused to a human Fc fragment to create a VHH2-Fc antibody that would offer prolonged serum persistence. After in planta (Nicotiana benthamiana) expression and purification, we show that our VHH2-Fc antibody retained high affinity binding to α–Cbtx. Mouse α–Cbtx challenge studies showed that our highest affinity VHHs (C2 and C20) and the VHH2-Fc antibody effectively neutralized lethality induced by α–Cbtx at an antibody:toxin molar ratio as low as ca. 0.75×:1. Further research towards the development of an antivenom therapeutic involving these anti-α-Cbtx VHHs and VHH2-Fc antibody molecules should involve testing them as a combination, to determine whether they maintain tissue penetration capability and low immunogenicity, and whether they exhibit improved serum persistence and therapeutic efficacy.
Maximizing the expression yields of recombinant whole antibodies and antibody fragments such as Fabs, single-chain Fvs and single-domain antibodies is highly desirable since it leads to lower production costs. Various eukaryotic and prokaryotic expression systems have been exploited to accommodate antibody expression but Escherichia coli systems have enjoyed popularity, in particular with respect to antibody fragments, because of their low cost and convenience. In many instances, product yields have been less than adequate and intrinsic and extrinsic variables have been investigated in an effort to improve yields. This review deals with various aspects of antibody expression in E. coli with a particular focus on single-domain antibodies.
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