Roles for fibrinogen, immunoglobulin and complement in platelet activation promoted by Staphylococcus aureus clumping factor A

Staphylococcus aureus is an important cause of infective endocarditis (IE) in patients without a history of prior heart valve damage. The ability to stimulate the activation of resting platelets and their subsequent aggregation is regarded as an important virulence factor of bacteria that cause IE. Clumping factor A is the dominant surface protein responsible for platelet activation by S. aureus cells in the stationary phase of growth. This study used Lactococcus lactis as a surrogate host to study the mechanism of ClfA-promoted platelet activation. Expression of ClfA from a nisin-inducible promoter demonstrated that a minimum level of surface-expressed ClfA was required. Using platelets that were purified from plasma, the requirement for both bound fibrinogen and immunoglobulin was demonstrated. The immunoglobulin G (IgG) requirement is consistent with the potent inhibition of platelet activation by a monoclonal antibody specific for the platelet FcgammaRIIa receptor. Furthermore the IgG must contain antibodies specific for the ClfA A domain. A model is proposed whereby bacterial cells armed with a sufficient number of surface-bound fibrinogen molecules can engage resting platelet glycoprotein GPIIb/IIIa, aided by bound IgG molecules, which encourages the clustering of FcgammaRIIa receptors. This can trigger activation of signal transduction leading to activation of GPIIb/IIIa and aggregation of platelets. In addition, analysis of a mutant of ClfA totally lacking the ability to bind fibrinogen revealed a second, although less efficient, mechanism of platelet activation. The fibrinogen-independent pathway required IgG and complement deposition to trigger platelet aggregation.     

A stereochemical study of the mechanism of activation of donor oligonucleotides by RNA ligase from bacteriophage T4 infected Escherichia coli

RNA ligase from bacteriophage T4 infected Escherichia coli catalyzes the activation of adenosine 3',5'-bisphosphate (representing the donor oligonucleotide) by adenosine 5'-[(S)-alpha-17O,alpha,alpha-18O2]triphosphate with retention of configuration at P alpha. Since single-step enzyme-catalyzed nucleotidyl transfer reactions proceed with inversion, this stereochemical result provides support for a double displacement mechanism involving an adenylyl-enzyme intermediate as proposed previously from isotope exchange experiments.     

Immunological comparison of microbial TPP-dependent non-oxidative α-keto acid decarboxylases

Abstract Antigenic, and hence possible evolutionary, relationships amongst various TPP-dependent non-oxidative α-keto acid decarboxylases were determined by the Ouchterlony double diffusion method and by measuring the degree of antibody-induced enzyme inhibition. The results show that: (a) phenylglyoxylate decarboxylases of various wild-type strains of Acinetobacter calcoaceticus are antigenically indistinguishable; (b) there seems to be no antigenic cross-reactivity between the phenylglyoxylate decarboxylase of A. calcoaceticus and of Pseudomonas aeruginosa or Pseudomonas putida ; and (c) no antigenic homology can be detected amongst phenylglyoxylate decarboxylase and phenylpyruvate decarboxylase of A. calcoaceticus and pyruvate decarboxylase of brewers' yeast.     

Diagnostic Potential of Zinc Finger Protein-Specific Autoantibodies and Associated Linear B-Cell Epitopes in Colorectal Cancer

Colorectal cancer is one of the most common cancers worldwide with almost 700,000 deaths every year. Detection of colorectal cancer at an early stage significantly improves patient survival. Cancer-specific autoantibodies found in sera of cancer patients can be used for pre-symptomatic detection of the disease. In this study we assess the zinc finger proteins ZNF346, ZNF638, ZNF700 and ZNF768 as capture antigens for the detection of autoantibodies in colorectal cancer. Sera from 96 patients with colorectal cancer and 35 control patients with no evidence of cancer on colonoscopy were analysed for the presence of ZNF-specific autoantibodies using an indirect ELISA. Autoantibodies to individual ZNF proteins were detected in 10–20% of colorectal cancer patients and in 0–5.7% of controls. A panel of all four ZNF proteins resulted in an assay specificity of 91.4% and sensitivity of 41.7% for the detection of cancer patients in a cohort of non-cancer controls and colorectal cancer patients. Clinicopathological and survival analysis revealed that ZNF autoantibodies were independent of disease stage and did not correlate with disease outcome. Since ZNF autoantibodies were shared between patients and corresponding ZNF proteins showed similarities in their zinc finger motifs, we performed an in silico epitope sequence analysis. Zinc finger proteins ZNF700 and ZNF768 showed the highest sequence similarity with a bl2seq score of 262 (E-value 1E-81) and their classical C2H2 ZNF motifs were identified as potential epitopes contributing to their elevated immunogenic potential. Our findings show an enhanced and specific immunogenicity to zinc finger proteins, thereby providing a multiplexed autoantibody assay for minimally invasive detection of colorectal cancer.     

Diatom species–environment relationships and inference models from Isachsen, Ellef Ringnes Island, Canadian High Arctic

Surface sediment diatom assemblages were examined from 26 freshwater sites near Isachsen (78°47N, 103°32W), Ellef Ringnes Island, a region of diverse and atypical water chemistry for high arctic sites. One hundred and sixty eight diatom taxa were identified from these samples, over 50% of which had not previously been recorded in the Canadian High Arctic. Variations in diatom assemblages were related to changes in measured environmental variables using multivariate techniques. Canonical correspondence analysis (CCA) indicated that five variables contributed significantly to explaining patterns of diatom variation (i.e., COND, DIC, Mn, TPF, TPU). The first CCA axis (=0.44) was primarily controlled by conductivity-related variables, while CCA axis 2 (=0.21) was related to particulate concentrations. Diatom-based inference models were generated for the reconstruction of conductivity (RMSEP_jack=0.32, r²_jack=0.76) and pH (RMSEP_jack=0.40, r²_jack=0.69). The strengths of these models indicate that it will be possible to reliably infer past trends in conductivity and pH from diatom assemblages preserved in dated sediment cores from the Isachsen region.     

Sub‐picomolar relaxin signalling by a pre‐assembled RXFP1, AKAP79, AC2, β‐arrestin 2, PDE4D3 complex

Biochemical studies suggest that G‐protein‐coupled receptors (GPCRs) achieve exquisite signalling specificity by forming selective complexes, termed signalosomes. Here, using cAMP biosensors in single cells, we uncover a pre‐assembled, constitutively active GPCR signalosome, that couples the relaxin receptor, relaxin family peptide receptor 1 (RXFP1), to cAMP following receptor stimulation with sub‐picomolar concentrations of peptide. The physiological effects of relaxin, a pleiotropic hormone with therapeutic potential in cancer metastasis and heart failure, are generally attributed to local production of the peptide, that occur in response to sub‐micromolar concentrations. The highly sensitive signalosome identified here provides a regulatory mechanism for the extremely low levels of relaxin that circulate. The signalosome includes requisite Gα_s, Gβγ and adenylyl cyclase 2 (AC2); AC2 is functionally coupled to RXFP1 through AKAP79 binding to helix 8 of the receptor; activation of AC2 is tonically opposed by protein kinase A (PKA)‐activated PDE4D3, scaffolded through a β‐arrestin 2 interaction with Ser⁷⁰⁴ of the receptor C‐terminus. This elaborate, pre‐assembled, ligand‐independent GPCR signalosome represents a new paradigm in GPCR signalling and provides a mechanism for the distal actions of low circulating levels of relaxin.     

In Vitro Measurement and Modeling of Platelet Adhesion on VWF-Coated Surfaces in Channel Flow

The process of platelet adhesion is initiated by glycoprotein (GP)Ib and GPIIbIIIa receptors on the platelet surface binding with von Willebrand factor on the vascular walls. This initial adhesion and detachment of a single platelet is a complex process that involves multiple bonds forming and breaking and is strongly influenced by the surrounding blood-flow environment. In addition to bond-level kinetics, external factors such as shear rate, hematocrit, and GPIb and GPIIbIIIa receptor densities have also been identified as influencing the platelet-level rate constants in separate studies, but this still leaves a gap in understanding between these two length scales. In this study, we investigate the fundamental relationship of the dynamics of platelet adhesion, including these interrelating factors, using a coherent strategy. We build a, to our knowledge, novel and computationally efficient multiscale model accounting for multibond kinetics and hydrodynamic effects due to the flow of a cellular suspension. The model predictions of platelet-level kinetics are verified by our microfluidic experiments, which systematically investigate the role of each external factor on platelet adhesion in an in vitro setting. We derive quantitative formulas describing how the rates of platelet adhesion, translocation, and detachment are defined by the molecular-level kinetic constants, the local platelet concentration near the reactive surface determined by red-blood-cell migration, the platelet effective reactive area due to its tumbling motion, and the platelet surface receptor density. Furthermore, if any of these aspects involved have abnormalities, e.g., in a disease condition, our findings also have clinical relevance in predicting the resulting change in the adhesion dynamics, which is essential to hemostasis and thrombosis.     

The Carbohydrate-linked Phosphorylcholine of the Parasitic Nematode Product ES-62 Modulates Complement Activation

Parasitic nematodes manufacture various carbohydrate-linked phosphorylcholine (PCh)-containing molecules, including ES-62, a protein with an N-linked glycan terminally substituted with PCh. The PCh component is biologically important because it is required for immunomodulatory effects. We showed that most ES-62 was bound to a single protein, C-reactive protein (CRP), in normal human serum, displaying a calcium-dependent, high-avidity interaction and ability to form large complexes. Unexpectedly, CRP binding to ES-62 failed to efficiently activate complement as far as the C3 convertase stage in comparison with PCh-BSA and PCh-containing Streptococcus pneumoniae cell wall polysaccharide. C1q capture assays demonstrated an ES-62-CRP-C1q interaction in serum. The three ligands all activated C1 and generated C4b to similar extents. However, a C2a active site was not generated following ES-62 binding to CRP, demonstrating that C2 cleavage was far less efficient for ES-62-containing complexes. We proposed that failure of C2 cleavage was due to the flexible nature of carbohydrate-bound PCh and that reduced proximity of the C1 complex was the reason that C2 was poorly cleaved. This was confirmed using synthetic analogues that were similar to ES-62 only in respect of having a flexible PCh. Furthermore, ES-62 was shown to deplete early complement components, such as the rate-limiting C4, following CRP interaction and thereby inhibit classical pathway activation. Thus, flexible PCh-glycan represents a novel mechanism for subversion of complement activation. These data illustrate the importance of the rate-limiting C4/C2 stage of complement activation and reveal a new addition to the repertoire of ES-62 immunomodulatory mechanisms with possible therapeutic applications.     

Quality improvement report: The “jaundice hotline” for the rapid assessment of patients with jaundice

ProblemPatients with jaundice require rapid diagnosis and treatment, yet such patients are often subject to delay.DesignAn open referral, rapid access jaundice clinic was established by reorganisation of existing services and without the need for significant extra resources.

Background and setting

A large general hospital in a largely rural and geographically isolated area.

Key measures for improvement

Waiting times for referral, consultation, diagnosis, and treatment, length of stay in hospital, and general practitioners'' and patients'' satisfaction with the service.

Strategies for change

Referrals were made through a 24 hour telephone answering machine and fax line. Initial assessment of patients was carried out by junior staff as part of their working week. Dedicated ultrasonography appointments were made available.

Effects of change

Of 107 patients seen in the first year of the service, 62 had biliary obstruction. The mean time between referral and consultation was 2.5 days. Patients who went on to endoscopic retrograde cholangiopancreatography waited 5.7 days on average. The mean length of stay in hospital in the 69 patients who were admitted was 6.1 days, compared with 11.5 days in 1996, as shown by audit data. Nearly all the 36 general practices (95%) and the 30 consecutive patients (97%) that were surveyed rated the service as above average or excellent.

Lessons learnt

An open referral, rapid access service for patients with jaundice can shorten time to diagnosis and treatment and length of stay in hospital. These improvements can occur through the reorganisation of existing services and with minimal extra cost.