J. Housden

A comparison of the wear and fatigue properties of plasma-assisted physical vapour deposition TiN, CrN and duplex coatings on Ti-6Al-4V

Abstract The study sets out to establish a comparison between duplex systems of plasma nitriding followed by plasma-assisted physical vapour deposition (PAPVD) of TiN deposited on Ti-6Al-4V, compared with PAPVD of TiN and CrN alone. The fatigue resistance has also been examined since conventional surface modifications can often impair fatigue resistance. A rubber-wheel-type abrasion tester and pin-on-disc sliding wear tester were used to examine the wear resistance of the coatings and the load-bearing capacity of the substrate respectively. A Wohler-type rotating tester was used to study the fatigue properties. Using smooth rotating-bending fatigue specimens tested in air at 5700 rev min-1 for 106 cycles or until failure it was found that TiN, CrN and the duplex coating did not impair fatigue resistance but actually improved the S-N curves and increased the endurance limit. From the wear results it was found that, although TiN and CrN do improve the wear resistance of Ti-6Al-4V significantly, it is the duplex coating that has much the greater load-bearing capacity on the titanium substrate and gives a significant improvement on PAPVD TiN or CrN coatings in sliding and abrasive wear conditions.

TiN and CrN PVD coatings on electroless nickel-coated steel substrates

Abstract Plasma-assisted (PA) PVD ceramic coating such as TiN have so far achieved only very limited use on cheap low-alloy steels, owing to problems relating to both corrosion resistance and the need for load support from the underlying material. Here we report tests to asses the wear and corrosion performance of TiN and CrN PAPVD coatings on phosphorus-doped electroless nickel (ENiP)-coated steels. It is shown that this route offers a potentially cost-effective means of utilizing PAPVD ceramic films on lower grade steels. In particular, CrN/ENiP on AISI 304 stainless steel is shown to exhibit a promising combination of wet abrasion resistance with good corrosion properties.

Substrate surface finish effects in duplex coatings of PAPVD TiN and CrN with electroless nickel-phosphorus interlayers

The ability to deposit hard coating materials on to a wide range of substrate surface finishes and still maintain adequate structural and functional properties is a very desirable objective in the quest to optimise product economics. A better surface finish requires longer and more elaborate finishing processes which will result in substantial additional costs. In this investigation two different groups of substrate surface roughnesses were prepared for a number of appropriate testing procedures. The tests involved in this study were microhardness, surface profilometry, scratch adhesion and the wet sand rubber wheel abrasive test. Electrochemical corrosion testing was also carried out. The results reveal that the substrate roughness can be optimised to achieve the best compromise in terms of cost and performance. The addition of an electroless nickel-phosphorus (ENiP) interlayer, in particular, is beneficial in isolating the substrate from near-surface effects and in permitting the use of rougher surface finishes.

The Importance of Lys-352 of Human Immunoglobulin E in FcϵRII/CD23 Recognition

The interaction of immunoglobulin E (IgE) with its low affinity receptor (FcϵRII/CD23) plays a central role in the initiation and regulation of type I hypersensitivity responses. We have previously identified the importance of amino acid residues in the A-B loop of the Cϵ3 domain of human IgE and implicated a region close to the glycosylation site at asparagine 371 as contributing to IgE-CD23 interaction. These residues were now targeted by site-directed mutagenesis. The IgE-CD23 interaction was assessed by semiquantitative flow cytometry. Replacement of the entire Cϵ3 A-B loop (residues 341–356) with the homologous rat IgE sequence resulted in complete loss of human CD23 recognition, as did replacement of residues 346–353, indicating that class-specific effector residue(s) are contained within these eight amino acids. Lysine 352 within the A-B loop was identified as contributing directly to human CD23 interaction. Mutation to the rodent homologue glycine or glutamate resulted in a significant reduction in binding compared with native IgE, whereas conservative substitution with arginine effected a small, but statistically significant, enhancement of CD23 binding. Mutation of the Cϵ3 glycosylation site at asparagine 371 to threonine or glutamine did not significantly affect CD23 recognition. Our results yield new insights into the structural basis of the hIgE-CD23 interaction and hold promise for the rational design of drugs that can manipulate IgE-mediated regulation of the allergic response.

Generation of canine-human Fc IgE chimeric antibodies for the determination of the canine IgE domain of interaction with FcɛRIα

Identification of the domain(s) of canine IgE that interact with Fc epsilon RI alpha may lead to novel therapeutic intervention strategies that inhibit the ability of canine IgE to engage Fc epsilon RI alpha. A panel of canine-human Fc IgE chimeric antibodies was constructed to investigate this interaction by replacing canine IgE-Fc domains with the corresponding human IgE-Fc domains since human IgE-Fc does not recognize canine Fc epsilon RI alpha. beta-Hexosaminidase release assays were performed to assess the ability of the chimeric antibodies to bind to and sensitize a novel RBL cell line transfected with canine Fc epsilon RI alpha for antigen induced mediator release. Replacing canine C epsilon2 with human C epsilon2 resulted in similar levels of release as those elicited by canine Fc IgE from RBL-2H3 cells transfected with either canine Fc epsilon RI alpha or human Fc epsilon RI alpha. Substitution of canine C epsilon4 with human C epsilon4 resulted in approximately 10% lower levels of release compared to cells sensitized with canine Fc IgE. Receptor binding by flow cytometry and cell activation could not be detected when transfected RBL cells were incubated with chimeric constructs where canine C epsilon2 and C epsilon4 were substituted with human C epsilon2 and C epsilon4. However, when this construct was incubated with cognate antigen prior to cell challenge mediator release was observed, albeit at a 20% lower level, indicating that while canine C epsilon3 is the only domain essential for binding to canine or human Fc epsilon RI alpha, species specific residues in canine Cepsilon2 and C epsilon4 inhibit dissociation of the ligand from the receptor.

Simple isolation method for the bulk isolation of wear particles from metal on metal bearing surfaces generated in a hip simulator test

Isolation and characterization of metal-on-metal (MoM) wear particles from simulator lubricants is essential to understand wear behaviour, ion release and associated corrosive activity related to the wear particles. Substantial challenges remain to establish a simple, precise and repeatable protocol for the isolation and analysis of wear particles due to their extremely small size, their tendency to agglomerate and degrade. In this paper, we describe a simple and efficient method for the bulk isolation and characterisation of wear particles from MoM bearings. Freeze drying was used to remove the large volume of water from the serum lubricant, enzymes used to digest the proteins and ultracentrifugation to finally isolate and purify the particles. The present study involved a total of eight steps for the isolation process and a wear particle extraction efficiency of 45% was achieved.

Assessing the role of Asp 194 in the transmembrane domains of the α-chain of the high-affinity receptor complex for immunoglobulin E in signal transduction.

The high-affinity receptor complex for IgE plays a pivotal role in allergic responses since cross-linking of the high-affinity IgE receptor (FcɛRI) on target cells initiates a signaling cascade facilitating release of inflammatory mediators causing allergic responses. The transmembrane regions of the ligand binding domains of the high-affinity IgE and low-affinity IgG receptors share an invariant motif (LFAVDTGL) containing a polar aspartate within a predominantly non-polar setting. The functional importance of this aspartate residue (D194) in FcɛRI-mediated receptor signaling was assessed by site-directed mutagenesis. Rat basophilic leukemia cells (RBL-2H3) transfected with the human IgE binding subunit (FcɛRIα) incorporating polar substitutions like asparagine (D194N) or threonine (D194T) resulted in the formation of a functional rat/human chimeric receptor complex. When activated via huIgE and antigen, cells transfected with these variant receptor subunits supported mediator release, intracellular calcium mobilisation and tyrosine phosphorylation of γ-chain and Syk kinase while a non-polar substitution (D194L) gave rise to cell surface expression of the mutated receptor subunit but failed to initiate downstream signaling. No cell surface expression of huFcɛRIα gene constructs was observed when D194 was replaced with the non-polar Ile (D194I) residue of similar size, the larger positively charged Arg (D194R) or lysine (D194K) residues, or the negatively charged glutamate (D194E) and smaller polar Ser (D194S) non-polar Ala (D194A) and V (D194V). These observations highlight importance of the size and charge of amino acid residue at position 194 in determining IgE receptor subunit interactions, cell surface localization, and initiation of downstream signaling events.

Identification of amino acid residues involved in the interaction of canine IgE with canine and human FcɛRIα

The interaction of immunoglobulin E (IgE) antibodies with the high-affinity receptor (FcεRI) is important in anti-parasitic immunity and plays a central role in allergic responses. It has been shown that the human Cε3 domains comprise the binding sites for FcεRIα and crystal structure determination has shown that amino acids in four sites contribute to the high affinity of the interaction. The role of homologous residues within canine IgE-Fc, i.e. amino acids located at Cε2-Cε3 interface (residues 332-337), loop BC (residues 362-365), loop DE (residues 393-396), and loop FG (residues 424-427) in canine Cε3 domain were targeted by site-specific mutagenesis. The functional consequences of the mutations to support (i) IgE-mediated, antigen-induced release of β-hexosaminidase from RBL cells transfected with canine or human FcεRIα and (ii) the affinity of the mutants for the soluble extracellular domain of the α-chain expressed in Pichia pastoris were determined by Surface Plasmon Resonance (SPR). Kinetic analysis supports the observed effects of IgE mutations on stimulus secretion coupling. Potential applications of this study, leading to the generation of an IgE variant with a disabled FcεRIα binding site, are discussed.

The role of Cɛ2, Cɛ3, and Cɛ4 domains in human and canine IgE and their contribution to FcɛRIα interaction

The Cε2 and Cε4 domains are considered as scaffolds, allowing Cε3 domains to assume an appropriate orientation to interact with FcεRI (Wurzburg and Jardetzky, 2002; Hunter et al., 2008). Human/canine IgE chimeric antibodies were expressed to assess the nature of the contribution of Cε2 and Cε4 domains to bind to and induce target cell degranulation via FcεRIα. Our results indicate that for (1) Cε3 domains in IgE of canine and human origin are the only necessary region for binding to FcεRIα. (2) The interaction of canine IgE with human sFcεRIα is significantly enhanced by contributions from both Cε2 and Cε4 domains of dog origin. (3) The canine/human IgE chimeric antibody construct rapidly dissociates from its the receptor when the canine Cε2 and Cε4 domains are replaced by the homologous human Fc domains which do not confer a conformation on the Cε3 domain to facilitate stable interaction with canine FcRIα. Kinetic constants for the binding of this chimera to the soluble extracellular domain of the receptor indicate an approximate 120-fold decrease in the affinity for canine sFcεRIα (ka=5.30 × 10(2)M(-1)s(-1)) and a 330-fold increase in the dissociation from canine sFcεRIα (KD=6.9 × 10(-6)M(-1)), compared to the wild type IgE kinetic constants (Ka=6.30 × 10(4)M(-1)s(-1); KD=2.1 × 10(-8)M(-1)). Although canine IgE does engage human FcεRIα, canine Cε2 and Cε4 do not contribute to the high-affinity of interaction with human FcεRIα. Upon replacement of human Cε2 and Cε4 domain by the canine homologues, human IgE Cε3 only retains a low affinity for the human receptor, which shows that Cε2 and Cε4 domains in human IgE Fc contribute significantly to the interaction with its cognate receptor.

Fc receptor-γ subunits with both polar or non-polar amino acids at position of T22 are capable of restoring surface expression of the high-affinity IgE receptor and degranulation in γ subunit-deficient rat basophilic leukemia cells.

The high-affinity IgE receptor (FcɛRI) is formed by the IgE-binding α subunit, β subunit and γ subunits homodimer. All three subunits are required for proper expression of the receptor on the plasma membrane of mast cells and basophils. However, the exact molecular mechanism of inter-subunit interactions required for correct expression and function of the FcɛRI complex remains to be identified. A recent study suggested that polar aspartate at position 194 within the transmembrane domain of the α subunit could interact by hydrogen bonding with polar threonine at position 22 in the transmembrane domains of the γ subunits. To verify this, we used previously isolated rat basophilic leukemia (RBL)-2H3 variant cells deficient in the expression of the FcɛRI-γ subunit (FcR-γ), and transfected them with DNA vectors coding for FcR-γ of the wild-type or mutants in which T22 was substituted for nonpolar alanine (T22A mutant) or polar serine (T22S mutant). Analysis of the transfectants showed that both T22A and T22S mutants were capable to restore surface expression of the FcɛRI similar to wild-type FcR-γ. Furthermore, cells transfected with wild-type, T22A or T22S FcR-γ showed comparably enhanced FcɛRI-mediated degranulation. Our data indicate that substitution of FcR-γ T22 with non-polar amino acid does not interfere with surface expression of the FcɛRI and its signaling capacity.