Total knee replacement survivorship by Design Philosophy: are we ignoring medial pivot design? Analysis based on the UK National Joint Registry.

The UK National Joint Registry(NJR) has not reported total knee replacement (TKR) survivorship based on design phi- losophy alone, unlike its international counterparts. We report outcomes of implant survivorship based on design phi- losophy using data from NJR's 2020 annual report. All TKR implants with an identifiable design philosophy from NJR data were included. Cumulative revision data for cruciate-retaining(CR), posterior stabilised(PS), mobile-bearing(MB) design philosophies was derived from merged NJR data. Cumulative revision data for individual brands of implants with the medial pivot (MP) philosophy were used to calculate overall survivorship for this design philosophy. The all-cause revision was used as the endpoint and calculated to 15 years follow-up with Kaplan-Meier curves. 1,144,384 TKRs were included. CR is the most popular design philosophy (67.4%), followed by PS(23.1%), MB (6.9%) and least commonly MP (2.6%). MP and CR implants showed the best survivorship (95.7% and 95.6% respectively) at 15 years which is statistically significant at, and beyond, 10 years. Observed survivorship was lower at all time points with the PS and MB implants (94.5% for both designs at 15 years). While all design philosophies considered in this study survive well, CR and MP designs offer statistically superior survivorship at and beyond 10 years. MP design performs better than CR beyond 13 years yet, remain the least popular design philosophy used. Publishing data based on knee arthroplasty design phi- losophy would help surgeons when making decisions on implant choice.

Chiari Malformation Presenting as Headaches Associated with Laughter.

Presentation We describe the case of a 5 year old boy, referred to our outpatient department with a one year history of headaches associated with laughter. Diagnosis Investigation with MRI Brain revealed Chiari Type 1 Malformation (CM-1), with cerebellar tonsillar descent of 19mm below the foramen magnum. Treatment He is being managed conservatively with serial neuroimaging and symptom monitoring. Discussion CM-1 is a hindbrain malformation characterised by ≥5mm herniation of the cerebellar tonsils.1,2 It is diagnosed radiographically, and is increasingly being detected incidentally.3,4 The natural history of asymptomatic patients is usually to remain asymptomatic, and symptomatic patients often show symptom improvement, particularly in paediatric populations.3,5 Neurosurgical interventions may be offered based on symptoms and radiographic findings, but carries a complication rate of 8.2%.6.

Evaluation of the role of platelet integrins in fibronectin-dependent spreading and adhesion.

BACKGROUND: Recent studies have shown that platelet adhesion and subsequent aggregation can occur in vivo in the absence of the two principal platelets adhesive ligands, von Willebrand factor and fibrinogen. These results highlight a possible role for fibronectin in supporting thrombus formation. OBJECTIVE AND METHODS: To evaluate the platelet integrins and subsequent activation pathways associated with fibronectin-dependent platelet adhesion utilizing both human and murine platelets. RESULTS: Platelets can adhere to fibronectin via the integrin alpha(IIb)beta(3), leading to formation of lamellipodia. This is mediated through an interaction with the tenth type III domain in fibronectin. Spreading on fibronectin promotes alpha(IIb)beta(3)-mediated Ca(2+) mobilization and tyrosine phosphorylation of focal adhesion kinase and phospholipase C gamma2. In contrast, studies with blocking antibodies and mice demonstrate that alpha(5)beta(1) and alpha(v)beta(3) support adhesion and promote formation of filopodia but not lamellipodia or tyrosine phosphorylation of these proteins. Further, neither alpha(5)beta(1) nor alpha(v)beta(3) is able to induce formation of lamellipodia in the presence of platelets agonists, such as collagen-related-peptide (CRP). CONCLUSIONS: These observations demonstrate that integrins alpha(5)beta(1) and alpha(v)beta(3) support platelet adhesion and the generation of filopodia but that, in contrast to the integrin alpha(IIb)beta(3), are unable to promote formation of lamellipodia.

Kinetics of LFA-1 binding to ICAM-1 studied in a cell-free system.

Neutrophil capture on inflamed endothelium is controlled by dynamic regulation of the integrin CD11a/CD18 (LFA-1). Small molecules, antibodies, and certain divalent cations binding to specific epitopes on the integrin are able to stabilize either a closed (low affinity) or open (high affinity) state. To determine the relationship between LFA-1 conformation and affinity for ICAM-1 we assembled a cell-free system consisting of CD11a/CD18 heterodimer adhered to latex microspheres. The kinetics of dimeric ICAM-1 binding to the LFA-1 on the microspheres was measured via flow cytometry and a real time conformational shift into a lower affinity state was observed by addition of a small molecule inhibitor.

CD18 activation epitopes induced by leukocyte activation.

The cell surface adhesion molecule LFA-1 coordinates leukocyte trafficking and is a costimulatory molecule for T cell activation. We developed a panel of mAbs that recognize activation epitopes on the CD18 subunit, and show that stimulation of T lymphocytes appears to be accompanied by a conformational change in a subpopulation of LFA-1 that does not require ligand binding. Activation epitope up-regulation requires divalent cations, is sensitive to cellular signal transduction events, and correlates with cell adhesion. In addition, the stimulated appearance of these activation epitopes is absent in cell lines from patients with leukocyte adhesion deficiency-1/variant that has previously been shown to be defective in LFA-1 activation. Thus, these activation epitope Abs can be used to dissect signal transmission to CD18. Evidence suggests that these CD18 activation epitopes are induced early in cellular activation and are independent of actin rearrangement necessary for avid adhesion. We have also determined that function-blocking CD18 Abs inhibit the induction of activation epitopes. One activation epitope Ab binds to a site on CD18 distinct from that of the blocking Abs, indicating that the blocking Abs suppress a conformational change in LFA-1. We also find that these neoepitopes are present on rLFA-1 with high affinity for ICAM-1 and their binding is modulated in parallel with the affinity of LFA-1 for ICAM-1. Collectively, these neoepitope Abs identify a subpopulation of LFA-1 most likely with high affinity for ICAM-1 and necessary for LFA-1 function.

Cellular activation of leukocyte function-associated antigen-1 and its affinity are regulated at the I domain allosteric site.

The I domain of the integrin LFA-1 possesses a ligand binding interface that includes the metal ion-dependent adhesion site. Binding of the LFA-1 ligand, ICAM-1 to the metal ion-dependent adhesion site is regulated by the I domain allosteric site (IDAS). We demonstrate here that intracellular signaling leading to activation of LFA-1 binding to ICAM-1 is regulated at the IDAS. Inhibitory mutations in or proximal to the IDAS are dominant to cytoplasmic signals that activate binding to ICAM-1. In addition, mutational activation at the IDAS greatly increases the binding of lymphocyte-expressed LFA-1 to ICAM-1 in response to PMA, but does not result in constitutive binding. Binding of a novel CD18 activation epitope mAb to LFA-1 in response to soluble ICAM-1 binding was also blocked by inhibitory and was enhanced by activating IDAS mutations. Surface plasmon resonance using soluble wild-type LFA-1 and an IDAS mutant of LFA-1 indicate that the IDAS can regulate a 6-fold change in the K(d) of ICAM-1 binding. The K(d) of wild-type LFA-1 (1.2 x 10(-1) s(-1)) differed with that of the activating IDAS mutant (1.9 x 10(-2) s(-1)), but their K(a) values were identical (2.2 x 10(5) M(-1)s(-1)). We propose that IDAS regulates the binding of LFA-1 to ICAM-1 activated by intracellular signals. IDAS can control the affinity state of LFA-1 with concomitant I domain and CD18 conformational changes.

The hairpin structure of the (6)F1(1)F2(2)F2 fragment from human fibronectin enhances gelatin binding.

The solution structure of the (6)F1(1)F2(2)F2 fragment from the gelatin-binding region of fibronectin has been determined (Protein Data Bank entry codes 1e88 and 1e8b). The structure reveals an extensive hydrophobic interface between the non-contiguous (6)F1 and (2)F2 modules. The buried surface area between (6)F1 and (2)F2 ( approximately 870 A(2)) is the largest intermodule interface seen in fibronectin to date. The dissection of (6)F1(1)F2(2)F2 into the (6)F1(1)F2 pair and (2)F2 results in near-complete loss of gelatin-binding activity. The hairpin topology of (6)F1(1)F2(2)F2 may facilitate intramolecular contact between the matrix assembly regions flanking the gelatin-binding domain. This is the first high-resolution study to reveal a compact, globular arrangement of modules in fibronectin. This arrangement is not consistent with the view that fibronectin is simply a linear 'string of beads'.

NMR and mutagenesis evidence for an I domain allosteric site that regulates lymphocyte function-associated antigen 1 ligand binding.

The leukocyte integrin, lymphocyte function-associated antigen 1 (LFA-1) (CD11a/CD18), mediates cell adhesion and signaling in inflammatory and immune responses. To support these functions, LFA-1 must convert from a resting to an activated state that avidly binds its ligands such as intercellular adhesion molecule 1 (ICAM-1). Biochemical and x-ray studies of the Mac-1 (CD11b/CD18) I domain suggest that integrin activation could involve a conformational change of the C-terminal alpha-helix. We report the use of NMR spectroscopy to identify CD11a I domain residues whose resonances are affected by binding to ICAM-1. We observed two distinct sites in the CD11a I domain that were affected. As expected from previous mutagenesis studies, a cluster of residues localized around the metal ion-dependent adhesion site (MIDAS) was severely perturbed on ICAM-1 binding. A second cluster of residues distal to the MIDAS that included the C-terminal alpha-helix of the CD11a I domain was also affected. Substitution of residues in the core of this second I domain site resulted in constitutively active LFA-1 binding to ICAM-1. Binding data indicates that none of the 20 substitution mutants we tested at this second site form an essential ICAM-1 binding interface. We also demonstrate that residues in the I domain linker sequences can regulate LFA-1 binding. These results indicate that LFA-1 binding to ICAM-1 is regulated by an I domain allosteric site (IDAS) and that this site is structurally linked to the MIDAS.

The leukocyte integrin alpha D beta 2 binds VCAM-1: evidence for a binding interface between I domain and VCAM-1.

The trafficking of leukocytes through tissues is supported by an interaction between the beta 2 (CD18) integrins CD11a/CD18 (LFA-1) and CD11b/CD18 (Mac-1) and their ligand ICAM-1. The most recently identified and fourth member of the beta 2 integrins, alpha D beta 2, selectively binds ICAM-3 and does not appear to bind ICAM-1. We have reported recently that alpha D beta 2 can support eosinophil adhesion to VCAM-1. Here we demonstrate that expression of alpha D beta 2 in a lymphoid cell that does not express alpha 4 integrins confers efficient binding to VCAM-1. In addition, a soluble form of alpha D beta 2 binds VCAM-1 with greater efficiency relative to ICAM-3. The I domain of alpha D contains a binding site for VCAM-1 since recombinant alpha D I domain binds specifically to VCAM-1. In addition, alpha D mAb that block cellular binding to VCAM-1 bind the alpha D I domain. Using VCAM-1 mutants we have determined that the binding site on VCAM-1 for alpha D beta 2 overlaps with that of alpha 4++ integrins. Substitution of VCAM-1 aspartate at position 40, D40, within the conserved integrin binding site, diminishes binding to alpha D beta 2 and abrogates binding to the alpha D I domain. The corresponding integrin binding site residue in ICAM-3 is also essential to alpha D beta 2 binding. Finally, we demonstrate that alpha D beta 2 can support lymphoid cell adhesion to VCAM-1 under flow conditions at levels equivalent to those mediated by alpha 4 beta 1. These results indicate that VCAM-1 can bind to an I domain and that the binding of alpha D beta 2 to VCAM-1 may contribute to the trafficking of a subpopulation of leukocytes that express alpha D beta 2.

alphadbeta2 integrin is expressed on human eosinophils and functions as an alternative ligand for vascular cell adhesion molecule 1 (VCAM-1).

The beta2 family of integrins, CD11a, CD11b, CD11c, and alphad, are expressed on most leukocytes. We show that the newest member of this family, alphad, is expressed on human eosinophils in peripheral blood, and surface expression can be upregulated within minutes by phorbol ester or calcium ionophore A23187. Culture of eosinophils with interleukin 5 (IL-5) leads to a two- to fourfold increase in alphad levels by 3-7 d without a change in alpha4 integrin expression. Eosinophils isolated from late phase bronchoalveolar lavage fluids express alphad at levels similar to that seen after 3 d of IL-5 culture. Regarding alphadbeta2 ligands, in both freshly isolated and IL-5-cultured eosinophils, as well as alphadbeta2-transfected Chinese hamster ovary cells, alphadbeta2 can function as a ligand for vascular cell adhesion molecule 1 (VCAM-1). This conclusion is based on the ability of monoclonal antibodies to alphad, beta2, or VCAM-1 to block cell attachment in static adhesion assays. In experiments with eosinophils, the relative contribution of alphadbeta2 integrin- mediated adhesion is enhanced after IL-5 culture. These experiments demonstrate that alphadbeta2 is an alternative ligand for VCAM-1, and this integrin may play a role in eosinophil adhesion to VCAM-1 in states of chronic inflammation.

NMR structure and mutagenesis of the N-terminal Dbl homology domain of the nucleotide exchange factor Trio.

Guanine nucleotide exchange factors for the Rho family of GTPases contain a Dbl homology (DH) domain responsible for catalysis and a pleckstrin homology (PH) domain whose function is unknown. Here we describe the solution structure of the N-terminal DH domain of Trio that catalyzes nucleotide exchange for Rac1. The all-alpha-helical protein has a very different structure compared to other exchange factors. Based on site-directed mutagenesis, functionally important residues of the DH domain were identified. They are all highly conserved and reside in close proximity on two a helices. In addition, we have discovered a unique capability of the PH domain to enhance nucleotide exchange in DH domain-containing proteins.

Solution structure of the cytohesin-1 (B2-1) Sec7 domain and its interaction with the GTPase ADP ribosylation factor 1.

Cytohesin-1 (B2-1) is a guanine nucleotide exchange factor for human ADP ribosylation factor (Arf) GTPases, which are important for vesicular protein trafficking and coatamer assembly in the cell. Cytohesin-1 also has been reported to promote cellular adhesion via binding to the beta2 integrin cytoplasmic domain. The solution structure of the Sec7 domain of cytohesin-1, which is responsible for both the protein's guanine nucleotide exchange factor function and beta2 integrin binding, was determined by NMR spectroscopy. The structure consists of 10 alpha-helices that form a unique tertiary fold. The binding between the Sec7 domain and a soluble, truncated version of human Arf-1 was investigated by examining 1H-15N and 1H-13C chemical shift changes between the native protein and the Sec7/Arf-1 complex. We show that the binding to Arf-1 occurs through a large surface on the C-terminal subdomain that is composed of both hydrophobic and polar residues. Structure-based mutational analysis of the cytohesin-1 Sec7 domain has been used to identify residues important for binding to Arf and for mediating nucleotide exchange. Investigations into the interaction between the Sec7 domain and the beta2 integrin cytoplasmic domain suggest that the two proteins do not interact in the solution phase.

Leupaxin is a novel LIM domain protein that forms a complex with PYK2.

We have identified a novel cytoplasmic protein, leupaxin, that is preferentially expressed in hematopoietic cells and is most homologous to the focal adhesion protein, paxillin. Leupaxin possesses two types of protein interaction domains. There are four carboxyl-terminal LIM domains in leupaxin that share 70% amino acid identity and 80% similarity with those in paxillin. Paxillin LIM domains mediate localization to focal contacts. In the amino-terminal region of leupaxin there are three short stretches of approximately 13 amino acids that share 70-90% similarity with paxillin LD motifs. Paxillin LD motifs have been implicated in focal adhesion kinase (FAK) and vinculin binding resulting in the localization of FAK to focal adhesions. Leupaxin is expressed in cell types, such as macrophage, that lack FAK. We demonstrate here that leupaxin associates with a second FAK family member, PYK2. As leupaxin and PYK2 are both preferentially expressed in leukocytes they may therefore form a cell type-specific signaling complex. We also demonstrate that leupaxin is a substrate for a tyrosine kinase in lymphoid cells and thus may function in and be regulated by tyrosine kinase activity. Leupaxin is thus a phosphotyrosine protein with LD and LIM binding motifs most homologous to paxillin that may assemble and regulate PYK2 signaling complexes in leukocytes.

Differential regulation of chemoattractant-stimulated beta 2, beta 3, and beta 7 integrin activity.

Leukocyte adhesion to endothelium and extravasation are dynamic processes that require activation of integrins. Chemoattractants such as IL-8 and FMLP are potent activators of leukocyte integrins. To compare the chemoattractant-stimulated activation of three integrins, alpha 4 beta 7, alpha L beta 2, and alpha V beta 3, in the same cellular context, we expressed an IL-8 receptor (IL-8RA) and FMLP receptor (FPR) in the lymphoid cell line JY. Chemoattractants induced a rapid increase in alpha L beta 2- and alpha V beta 3-dependent JY adhesion within 5 min, and it was sustained for 30 min. In contrast, stimulation of alpha 4 beta 7-dependent adhesion was transient, returning to basal levels by 30 min. The activation profiles of the integrins were similar regardless of whether IL-8 or FMLP was used for induction. We also demonstrate that alpha 4 beta 7-dependent adhesion was uniquely responsive to the F actin-disrupting agent cytochalasin D and the protein kinase C (PKC) inhibitor chelerythrin. While alpha V beta 3- and alpha L beta 2-mediated cell adhesion was significantly reduced by cytochalasin D, alpha 4 beta 7-mediated adhesion was enhanced. Chelerythrin inhibited both the IL-8 and PMA activation of alpha L beta 2 and alpha V beta 3. In contrast, inducible alpha 4 beta 7 activity was unaffected, and basal activity was increased. These findings demonstrate that the mechanism of alpha 4 beta 7 regulation by chemoattractants is different from that of alpha L beta 2 and alpha V beta 3 and that it appears to involve distinct cytoskeletal and PKC dependencies. In addition, PKC activity may be a positive or negative regulator of integrin-dependent adhesion.

Crystal structure of a cytokine-binding region of gp130.

The structure of the cytokine-binding homology region of the cell surface receptor gp130 has been determined by X-ray crystallography at 2.0 A resolution. The beta sandwich structure of the two domains conforms to the topology of the cytokine receptor superfamily. This first structure of an uncomplexed receptor exhibits a similar L-shaped quaternary structure to that of ligand-bound family members and suggests a limited flexibility in relative domain orientation of some 3 degrees. The putative ligand-binding loops are relatively rigid, with a phenylalanine side chain similarly positioned to exposed aromatic residues implicated in ligand binding for other such receptors. The positioning and structure of the N-terminal portion of the polypeptide chain have implications for the structure and function of cytokine receptors, such as gp130, which contain an additional N-terminal immunoglobulin-like domain.

The interactions of the cytokine-binding homology region and immunoglobulin-like domains of gp130 with oncostatin M: implications for receptor complex formation.

The receptor gp130 is utilized by cytokines including interleukin 6, leukemia inhibitory factor, oncostatin M, cilary neurotrophic factor and cardiotrophin. It is essential for myocardial development and haematopoiesis during embryogenesis, and its role as a shared signal transducer among different cytokines explains their overlapping biological functions. Although gp130 contains a cytokine-binding homology region (CHR) analogous to the extracellular growth hormone receptor, the complexes that utilize gp130 are not simple dimerizations of receptors around a single cytokine but involve receptor interactions with additional sites on the ligand resulting in higher order complexes. Analysis by surface plasmon resonance of the binding of the immunoglobulin-like and CHR domains of the extracellular portion of gp130 to mutants of the cytokine oncostatin M reveal that the CHR forms the main binding site for oncostatin M by a classical site II interaction, but in addition a second interaction occurs involving the receptor's immunoglobulin-like domain and the cytokine's site III at the N-terminus of the D helix. The implications for complex formation are discussed.

L-selectin crosslinking induces integrin-dependent adhesion: evidence for a signaling pathway involving PTK but not PKC.

L-selectin mediates the initial contact of leukocytes with the endothelium prior to extravasation. Here we demonstrate that L-selectin engagement can induce rapid and avid integrin-dependent T cell adhesion to recombinant immobilized cell adhesion molecules (CAMs) including ICAM-1, ICAM-3, and VCAM-1, as well as to the extracellular matrix protein fibronectin (FN). L-selectin-induced adhesion to these integrin ligands shares characteristics with CD3 mAb- or phorbol ester-induced adhesion in requiring metabolic energy, tyrosine kinase and ligand-stimulated Ca2+ channel activity. However, L-selectin-induced adhesion is distinct from that induced by phorbol ester or CD3 crosslinking in being relatively independent of protein kinase C (PKC) activity and actin polymerization. Consistent with the higher levels of L-selectin expression on CD45RA+ (naive) cells, L-selectin crosslinking induces a greater proportion of naive relative to memory cell binding to CAMs and FN. In contrast, exposure to phorbol ester or CD3 crosslinking is more effective in inducing CD45RO+ (memory) cell adhesion. Thus, in addition to its role in leukocyte capture and rolling on the endothelium, L-selectin may contribute to beta 1 and beta 2 integrin-dependent binding and arrest.

Biotinylated lipopolysaccharide binds to endotoxin receptor in endothelial and monocytic cells.

Endotoxin or lipopolysaccharide (LPS), a major cell surface component of gram-negative bacteria, which could bind to different cell types when released into the bloodstream, plays a central role in the pathogenesis of septic shock syndromes. We have established a biotinylation procedure for labeling purified LPS molecules from Salmonella minnesota R595 and Escherichia coli J5 bacteria. The biotin group was conjugated to the bacterial LPS either by chemical oxidation of the LPS carbohydrate moiety (inner core region), followed by reduction with biotin-LC-hydrazide (biotinamido hexanoyl hydrazide), or by photoactivatable cross-linking with biotin-LC-ASA [1-(4-azidosalicylamido-)-6(biotinamido)-hexane], which was randomly attached to the carbohydrate and fatty acid (lipid A) groups of the LPS. Both labeled products retained biological activity (or endotoxicity) as evidenced by coagulation of the Limulus amoebocyte lysate. To determine its ability to bind avidin/streptavidin which in turn could be conjugated with enzymatic and fluorescent probes, the biotinylated LPS was used in enzyme immunoassay, Western blot, and flow cytometry. These assays were also used to analyze the binding of LPS ligand to its counterreceptor(s) on whole cell surface, membrane fragments, and in detergent lysates from human endothelial and monocytic cells. The described biotinylated LPS probes can be applied in a wide variety of techniques in receptor biochemistry, immunohistochemistry, and molecular cell biology.

A novel leukointegrin, alpha d beta 2, binds preferentially to ICAM-3.

The leukocyte-restricted beta 2 (CD18) integrins mediate cell adhesion in a variety of events essential for normal immune function. Despite extensive research in this field, only three members of this integrin subfamily have been described: CD11a/CD18 (LFA-1), CD11b/CD18 (Mac-1), and CD11c/CD18 (p150,95). We have identified a cDNA encoding a fourth alpha chain, alpha d, that associates with CD18. The alpha d subunit is more closely related to CD11b and CD11c than to CD11a. This integrin is expressed at moderate levels on myelomonocytic cell lines and subsets of peripheral blood leukocytes, and more strongly on tissue-compartmentalized cells such as foam cells, specialized macrophages found in aortic fatty streaks that may develop into atherosclerotic lesions. The alpha d/CD18 molecule exhibits preferential recognition of ICAM-3 over ICAM-1.