Biomarkers of exercise-induced myocardial stress in relation to inflammatory and oxidative stress

Increased concentrations of biomarkers reflecting myocardial stress such as cardiac troponin I and T and brain natriuretic peptide (BNP) have been observed following strenuous, long-lasting endurance exercise. The pathophysiological mechanisms are still not fully elucidated and the interpretations of increased post-exercise concentrations range from (i) evidence for exercise-induced myocardial damage to (ii) non-relevant spurious troponin elevations, presumably caused by assay imprecision or heterophilic antibodies. Several lines of evidence suggest that inflammatory processes or oxidative stress could be involved in the rise of NT-proBNP and Troponin observed in critically ill patients with sepsis or burn injury. We tested the hypothesis that inflammatory or oxidative stress is also responsible for exercise-induced cardiomyocyte strain in a large cohort of triathletes following an Ironman triathlon. However, the post-race increase in cardiac troponin T and NT-proBNP was not associated with several markers of exercise-induced inflammation, oxidative stress or antioxidant vitamins. Therefore, we clearly need more studies with other inflammatory markers and different designs to elucidate the scientific background for increases in myocardial stress markers following strenuous endurance events.

Serum levels of soluble receptor for advanced glycation end products and of S100 proteins are associated with inflammatory, autoantibody, and classical risk markers of joint and vascular damage in rheumatoid arthritis

Introduction: The receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily of cell surface receptor molecules. High concentrations of three of its putative proinflammatory ligands, S100A8/A9 complex (calprotectin), S100A8, and S100A12, are found in rheumatoid arthritis (RA) serum and synovial fluid. In contrast, soluble RAGE (sRAGE) may prevent proinflammatory effects by acting as a decoy. This study evaluated the serum levels of S100A9, S100A8, S100A12 and sRAGE in RA patients, to determine their relationship to inflammation and joint and vascular damage. Methods: Serum sRAGE, S100A9, S100A8 and S100A12 levels from 138 patients with established RA and 44 healthy controls were measured by ELISA and compared by unpaired t test. In RA patients, associations with disease activity and severity variables were analyzed by simple and multiple linear regressions. Results: Serum S100A9, S100A8 and S100A12 levels were correlated in RA patients. S100A9 levels were associated with body mass index (BMI), and with serum levels of S100A8 and S100A12. S100A8 levels were associated with serum levels of S100A9, presence of anti-citrullinated peptide antibodies (ACPA), and rheumatoid factor (RF). S100A12 levels were associated with presence of ACPA, history of diabetes, and serum S100A9 levels. sRAGE levels were negatively associated with serum levels of C-reactive protein (CRP) and high-density lipoprotein (HDL), history of vasculitis, and the presence of the RAGE 82Ser polymorphism. Conclusions: sRAGE and S100 proteins were associated not just with RA inflammation and autoantibody production, but also with classical vascular risk factors for end-organ damage. Consistent with its role as a RAGE decoy molecule, sRAGE had the opposite effects to S100 proteins in that S100 proteins were associated with autoantibodies and vascular risk, whereas sRAGE was associated with protection against joint and vascular damage. These data suggest that RAGE activity influences co-development of joint and vascular disease in rheumatoid arthritis patients.

Allergen capture by IgE and IgG enhanced cell-binding by allergen multimers: How complex is it?

Allergic diseases are the most common chronic disease of the western world, costing $7.8 billion per year in lost productivity and medical care in Australia alone.1 IgE is central to the immunopathogenesis of allergic diseases and important advances are now being made on multiple fronts of IgE research. In particular, two groups independently invested in the generation of IgE reporter mice to address the vexing question of the route of development of the elusive IgE+ B cell.2, 3 Two new anti-IgE mAb targeting membrane IgE and cell-bound IgE have the potential to deplete the cellular source of IgE.4, 5 These could be candidates for alternative anti-IgE treatment options with advantages over current anti-IgE therapy (OmalizumAb), which depletes free serum IgE. Researchers are still intrigued by the modes of interaction of IgE with allergen, and with both its receptors; the high affinity FcεR1 on mast cells and basophils, and the low affinity, C-type lectin, IgE receptor, CD23,6 on B cells and monocytes (Figure 1a and b). A new approach to the study of the complexity of these interactions was recently reported by Reginald et al.7 on page 167 of this issue.

Purification of the major group 1 allergen from bahia grass pollen, Pas n 1

Background Group 1 grass pollen allergens are glycoproteins of the β-expansin family. They are a predominant component of pollen and are potent allergens with a high frequency of serum IgE reactivity in grass pollen-allergic patients. Bahia grass is distinct from temperate grasses and has a prolonged pollination period and wide distribution in warmer climates. Here we describe the purification of the group 1 pollen allergen, Pas n 1, from Bahia grass (Paspalum notatum), an important subtropical aeroallergen source. Methods Pas n 1 was purified from an aqueous Bahia grass pollen extract by ammonium sulphate precipitation, hydrophobic interaction and size exclusion chromatography, and assessed by one- and two-dimensional gel electrophoresis, immunoblotting and ELISA. Results Pas n 1 was purified to a single 29-kDa protein band containing two dominant isoforms detected by an allergen-specific monoclonal antibody and serum IgE of a Bahia grass pollen-allergic donor. The frequency of serum IgE reactivity with purified Pas n 1 in 51 Bahia grass pollen-allergic patients was 90.6%. Serum IgE reactivity with purified Pas n 1 was highly correlated with serum IgE reactivity with Bahia grass pollen extract and recombinant Pas n 1 (r = 0.821 and 0.913, respectively). Conclusions Pas n 1 is a major allergen reactive at high frequency with serum IgE of Bahia grass pollen-allergic patients. Purified natural Pas n 1 has utility for improved specific diagnosis and immunotherapy for Bahia grass pollen allergy.

The dominant 55kDa allergen of the subtropical Bahia grass (Paspalum notatum) pollen is a group 13 pollen allergen, Pas n 13

Bahia grass, Paspalum notatum, is an important pollen allergen source with a long season of pollination and wide distribution in subtropical and temperate regions. We aimed to characterize the 55. kDa allergen of Bahia grass pollen (BaGP) and ascertain its clinical importance. BaGP extract was separated by 2D-PAGE and immunoblotted with serum IgE of a grass pollen-allergic patient. The amino-terminal protein sequence of the predominant allergen isoform at 55. kDa had similarity with the group 13 allergens of Timothy grass and maize pollen, Phl p 13 and Zea m 13. Four sequences obtained by rapid amplification of the allergen cDNA ends represented multiple isoforms of Pas n 13. The predicted full length cDNA for Pas n 13 encoded a 423 amino acid glycoprotein including a signal peptide of 28 residues and with a predicted pI of 7.0. Tandem mass spectrometry of tryptic peptides of 2D gel spots identified peptides specific to the deduced amino acid sequence for each of the four Pas n 13 cDNA, representing 47% of the predicted mature protein sequence of Pas n 13. There was 80.6% and 72.6% amino acid identity with Zea m 13 and Phl p 13, respectively. Reactivity with a Phl p 13-specific monoclonal antibody AF6 supported designation of this allergen as Pas n 13. The allergen was purified from BaGP extract by ammonium sulphate precipitation, hydrophobic interaction and size exclusion chromatography. Purified Pas n 13 reacted with serum IgE of 34 of 71 (48%) grass pollen-allergic patients and specifically inhibited IgE reactivity with the 55. kDa band of BaGP for two grass pollen-allergic donors. Four isoforms of Pas n 13 from pI 6.3-7.8 had IgE-reactivity with grass pollen allergic sera. The allergenic activity of purified Pas n 13 was demonstrated by activation of basophils from whole blood of three grass pollen-allergic donors tested but not control donors. Pas n 13 is thus a clinically relevant pollen allergen of the subtropical Bahia grass likely to be important in eliciting seasonal allergic rhinitis and asthma in grass pollen-allergic patients.

An arthritogenic alphavirus uses the α1β1 integrin collagen receptor

Ross River (RR) virus is an alphavirus endemic to Australia and New Guinea and is the aetiological agent of epidemic polyarthritis or RR virus disease. Here we provide evidence that RR virus uses the collagen-binding α1β1 integrin as a cellular receptor. Infection could be inhibited by collagen IV and antibodies specific for the β1 and α1 integrin proteins, and fibroblasts from α1-integrin-/- mice were less efficiently infected than wild-type fibroblasts. Soluble α1β1 integrin bound immobilized RR virus, and peptides representing the α1β1 integrin binding-site on collagen IV inhibited virus binding to cells. We speculate that two highly conserved regions within the cell-receptor binding domain of E2 mimic collagen and provide access to cellular collagen-binding receptors.

Bahia grass pollen specific IgE is common in seasonal rhinitis patients but has limited cross-reactivity with Ryegrass

Background: Perennial Ryegrass is a major cause of rhinitis in spring and early summer. Bahia grass, Paspalum notatum, flowers late into summer and could account for allergic rhinitis at this time. We determined the frequency of serum immunoglobulin (Ig)E reactivity with Bahia grass in Ryegrass pollen allergic patients and investigated IgE cross-reactivity between Bahia and Ryegrass. Methods: Serum from 33 Ryegrass pollen allergic patients and 12 nonatopic donors were tested for IgE reactivity with Bahia and Ryegrass pollen extracts (PE) by enzyme-linked immunosorbent assay (ELISA), western blotting and inhibition ELISA. Allergen-specific antibodies from a pool of sera from allergic donors were affinity purified and tested for IgE cross-reactivity. Results: Seventy-eight per cent of the sera had IgE reactivity with Bahia grass, but more weakly than with Ryegrass. Antibodies eluted from the major Ryegrass pollen allergens, Lol p 1 and Lol p 5, showed IgE reactivity with allergens of Ryegrass and Canary but not Bahia or Bermuda grasses. Timothy, Canary and Ryegrass inhibited IgE reactivity with Ryegrass and Bahia grass, whereas Bahia, Johnson and Bermuda grass did not inhibit IgE reactivity with Ryegrass. Conclusions: The majority of Ryegrass allergic patients also showed serum IgE reactivity with Bahia grass PE. However, Bahia grass and Ryegrass had only limited IgE cross-reactivity indicating that Bahia grass should be considered in diagnosis and treatment of patients with hay fever late in' the grass pollen season.

Use of phage display technology to investigate allergen-antibody interactions

Phage display is an advanced technology that can be used to characterize the interactions of antibody with antigen at the molecular level. It provides valuable data when applied to the investigation of IgE interaction with allergens. The aim of this rostrum article is to provide an explanation of the potential of phage display for increasing the understanding of allergen- IgE interaction, the discovery of diagnostic reagents, and the development of novel therapeutics for the treatment of allergic disease. The significance of initial studies that have applied phage display technology in allergy research will be highlighted. Phage display has been used to clone human IgE to timothy grass pollen allergen Phl p 5, to characterize the epitopes for murine and human antibodies to a birch pollen allergen Bet v 1, and to elucidate the epitopes of a murine mAb to the house dust mite allergen Der p 1. The technology has identified peptides that functionally mimic sites of human IgE constant domains and that were used to raise antiserum for blocking binding of IgE to the FcεRI on basophils and subsequent release of histamine. Phage display has also been used to characterize novel peanut and fungal allergens. The method has been used to increase our understanding of the molecular basis of allergen-IgE interactions and to develop clinically relevant reagents with the pharmacologic potential to block the effector phase of allergic reactions. Many advances from these early studies are likely as phage display technology evolves and allergists gain expertise in its research applications.

Antibody reactivity to the transmembrane protein of the caprine arthritis encephalitis virus correlates with severity of arthritis: No evidence for the involvement of epitope mimicry

Serum and synovial antibody reactivities of caprine arthritis encephalitis virus (CAEV) infected goats were assessed by Western blotting against purified CAEV antigen and the greatest intensity of reactivity in the serum of arthritic goats was to the gp45 transmembrane protein (TM). The extracytoplasmic domain of the TM gene was cloned into a pGEX vector and expressed in Escherichia coil as a glutathione S transferase fusion protein (GST-TM). This clone was found to be 90.5 and 89.2% homologous to published sequences of CAEV TM gene. Serum of 16 goats naturally infected with CAEV were examined by Western blotting for reactivity to the fusion protein. Antibody reactivity to the GST-TM correlated with clinically detectable arthritis (R = 0.642, P ≤ 0.007). The hypothesis that the immune response to the envelope proteins of the CAEV contributes to the severity of arthritis in goats naturally infected with CAEV via epitope mimicry was tested. Antibodies from 5 CAEV infected goats were affinity purified against the GST-TM fusion protein and tested for cross-reactivity with a series of goat synovial extracts and proteogylcans. No serum antibody response or cross-reactivity of affinity purified antibodies could be detected. Peptides of the CAEV SU that were predicted to be linear epitopes and a similar heat shock protein 83 (HSP) peptide identified by database searching, were synthesized and tested for reactivity in CAEV goats using ELISA, in vitro lymphocyte proliferation and delayed type hypersensitivity (DTH) assays. Peripheral blood lymphocytes from 10 of 17 goats with long term natural CAEV infections proliferated in vitro in response to CAEV and in vivo 3 of 7 CAEV infected goats had a DTH reaction to CAEV antigen. However, none of the peptides elicited significant cell mediated immune responses from CAEV infected goats. No antibody reactivity to the SU peptides or HSP peptide was found. We observed that the antibody reactivity to the CAEV TM protein associated with severity of arthritis however epitope mimicry by the envelope proteins of CAEV is unlikely to be involved.

Mimicry of 21. 5 KDa myelin basic protein peptide by a Maedi Visna virus polymerase peptide does not contribute to the pathogenesis of encephalitis in sheep

Epitope mimicry is the theory that an infectious agent such as a virus causes pathological effects via mimicry of host proteins and thus elicits a cross-reactive immune response to host tissues. Weise and Carnegie (1988) found a region of sequence similarity between the pol gene of the Maedi Visna virus (MVV), which induces demyelinating encephalitis in sheep, and myelin basic protein (MBP), which is known to induce experimental allergic encephalitis (EAE) in laboratory animals. In this study, cross-reactions between sera raised in sheep against synthetic peptides of MVV (TGKIPWILLPGR) and 21.5 kDa MBP (SGKVPWLKRPGR) were demonstrated using enzyme-linked immunosorbant assay (ELISA) and thin layer chromatography (TLC) immunoprobing. The antibody responses of MVV-infected sheep were investigated using ELISA against the peptides, and MBP protein, immunoprobing of the peptides on TPC plates and Western blotting against MBP. Slight significant reactions to the 21.5 kDa MBP peptide (P < 0.001) and to a lesser extent sheep MBP (P < 0.004) were detected in ELISA. The MBP peptide evoked stronger responses from more sera than the MVV peptide on immunoprobed TLC plates. On the Western blots, eight of the 23 sheep with Visna had serum reactivity to MBP. This slight reaction to MBP in MVV-infected sheep is of interest because of the immune responses to MBP evident in multiple sclerosis and EAE, but its relevance in Visna is limited since no correlation with disease severity was observed. The cell-mediated immune responses of MVV-infected sheep against similar peptides was assessed. The peptides did not stimulate proliferation of peripheral blood lymphocytes of MVV-infected sheep. Since the MVV peptide was not recognised by antibodies or T lymphocytes from MVV-infected and encephalic sheep, it was concluded that epitope mimicry of this 21.5 kDa MBP peptide by the similar MVV pol peptide was not contributing to the immunopathogenesis of Visna. The slight antibody response to MBP and the MBP peptide can be attributed to by-stander effects of the immunopathology of MVV-induced encephalitis.

Genetic and genomic studies of PADI4 in rheumatoid arthritis

Objectives. Strong genetic association of rheumatoid arthritis (RA) with PADI4 (peptidyl arginine deiminase) has previously been described in Japanese, although this was not confirmed in a subsequent study in the UK. We therefore undertook a further study of genetic association between PADI4 and RA in UK Caucasians and also studied expression of PADI4 in the peripheral blood of patients with RA. Methods. Seven single-nucleotide polymorphisms (SNP) were genotyped using polymerase chain reaction (PCR)-restriction fragment length polymorphism in 111 RA cases and controls. A marker significantly associated with RA (PADI4_100, rs#2240339) in this first data set (P = 0.03) was then tested for association in a larger group of 439 RA patients and 428 controls. PADI4 transcription was also assessed by real-time quantitative PCR using RNA extracted from peripheral blood mononuclear cells from 13 RA patients and 11 healthy controls. Results. A single SNP was weakly associated with RA (P = 0.03) in the initial case-control study, a single SNP (PADI4_100) and a two marker haplotype of that SNP and the neighbouring SNP (PADI4_04) were significantly associated with RA (P = 0.02 and P = 0.03 respectively). PADI4_100 was not associated with RA in a second sample set. PADI4 expression was four times greater in cases than controls (P = 0.004), but expression levels did not correlate with the levels of markers of inflammation. Conclusion. PADI4 is significantly overexpressed in the blood of RA patients but genetic variation within PADI4 is not a major risk factor for RA in Caucasians.

Immunization of male rabbits with sheep luteal receptor to LH results in production of antibodies exhibiting hormone-agonistic and -antagonistic activities

Antibodies to LH/chorionic gonadotrophin receptor (LH/CG-R; molecular weight 67 000), isolated in a homogenous state (established by SDS-PAGE and ligand blotting) from sheep luteal membrane using human CG (hCG)-Sepharose affinity chromatography, were raised in three adult male rabbits (R-I, R-II and R-III). Each of the rabbits received 20-30 mu g oi the purified receptor in Freund's complete adjuvant at a time. Primary immunization was followed by booster injection at intervals. Production of receptor antibodies was monitored by (1) determining the dilution of the serum (IgG fraction) that could specifically bind 50% of I-125-LH/CG-R added and (2) analysing sera for any chance in testosterone levels. Following primary immunization and the first booster, all three rabbits exhibited a 2.5- to 6.0-fold increase in serum testosterone over basal levels and this effect was spread over a period of time (similar to 40 days) coinciding with the rise and fall of receptor antibodies. The maximal antibody titre (ED(50)) produced at this time ranged from 1:350 to 1:100 to below detectable limits for R-I, R-II and R-III respectively. Subsequent immunizations followed by the second booster resulted in a substantial increase in antibody titre (ED(50) of 1:5000) in R-I, but this was not accompanied by any change in serum testosterone over preimmune levels, suggesting that with the progress of immunization the character of the antibody produced had also changed. Two pools of antisera from R-I collected 10 days following the booster (at day 70 (bleed I) and day 290 (bleed II)) were used in further experiments. IgG isolated from bleed I but not from bleed II antiserum showed a dose-dependent stimulation of testosterone production by mouse Leydig cells in vitro, thus confirming the in vivo hormone-mimicking activity antibodies generated during the early immunization phase. The IgG fractions from both bleeds were, however, capable of inhibiting (1) I-125-hCG binding to crude sheep luteal membrane (EC(50) of 1:70 and 1:350 for bleed I and II antisera respectively) and (2) ovine LH-stimulated testosterone production by mouse Leydig cells in vitro, indicating the presence oi antagonistic antibodies irrespective of the period of time during which the rabbits were immunized. The: fact that bleed I-stimulated testosterone production could be inhibited in a dose-dependent manner by the addition of IgG from bleed II to the mouse Leydig cell in vitro assay system showed that the agonistic activity is intrinsic to the bleed I antibody. The receptor antibody (bleed II) was also capable of blocking LH action in vivo, as rabbits passively (for 24 h with LH/CG-R antiserum) as well as actively (for 130 days) immunized against LH/CG-R failed to respond to a bolus injection of LH (50 mu g). At no time, however, was the serum testosterone reduced below the basal level. This study clearly shows that, unlike with LH antibody, attempts to achieve an LH deficiency effect in vivo by resorting to immunization with hole LH receptor is difficult, as receptor antibodies exhibit both hormone-mimicking (agonistic) as well as hormone-blocking (antagonistic) activities.

Expression of the extracellular domain of the human heat-stable enterotoxin receptor in Escherichia coli and generation of neutralizing antibodies

The entire extracellular domain of the human heat-stable enterotoxin (ST) receptor as well as a truncated N-terminal domain were cloned as glutathione S-transferase fusion proteins and expressed in Escherichia coli. The recombinant fusion proteins were purified from both the cytosol and the inclusion body fractions by selective detergent extraction followed by glutathione-agarose affinity chromatography. The purified protein, corresponding to the entire extracellular domain, bound the stable toxin peptide with an affinity comparable to that of the native receptor characterized from the human colonic T84 cell line. No binding was observed with the N-terminal truncated fragment of the receptor under similar conditions, Polyclonal antibodies were raised to the entire extracellular domain fusion protein as well as the truncated extracellular domain fusion protein, and the antibodies were purified by affinity chromatography. Addition of the purified antibodies to T84 cells inhibited ST binding and abolished ST-mediated cGMP production, indicating that critical epitopes involved in ligand interaction are present in the N-terminal fragment of the receptor, Purified antibodies recognized a single protein of M(r) 160,000 Da on Western blotting with T84 membranes, corresponding to a size of the native glycosylated receptor in T84 cells. These studies are the first report of the expression, purification, and characterization of any member of the guanylyl cyclase family of receptors in E. coli and show that binding of the toxin to the extracellular domain of the receptor is possible in the absence of any posttranslational modifications such as glycosylation. The recombinant fusion proteins as well as the antibodies that we have generated could serve as useful tools in the identification of critical residues of the extracellular domain involved in ligand interaction.

Antibodies to guanosine: Fractionation and specificities

Bovine serum albumin conjugates of guanosine prepared by the periodate method was used as immunogen to elicit guanosine antibodies in rabbits. The specificities of the antibodies were studied by the inhibition of their binding to [3H]Gox-red, [32P]DNA and [3H]RNA by related non-radioactive compounds. A population of antibodies is specific to Gox-red with an average association constant of around 107M−1 at 4°C. There are a population of antibodies which bind to [32P]ssDNA and [3H]RNA specifically at guanosine residues. RNA binding antibodies were separated into two populations by affinity chromatography.

Base specific binding of deoxyguanylate and deoxycytidylate antibodies to double stranded DNA

Antibodies raised in rabbits against daoxyguanylate and daoxycytidylate bind to 3H-(lambda) double stranded DNA and the binding is base specific. The concentrations of antibody populations that bind to double stranded DNA are much less than those binding to denatured DNA. Due to their low concentrations, these antibodies ware not detected in earlier studies. These antibodies are expected to be useful to probe the conformational flexibilities of double stranded DNAs.