Dissociation between murine spleen cell mitogenic activity of enterotoxin contaminants and anti-tumor activity of staphylococcal protein A

Soluble staphylococcal protein A (SpA) in the form of high m.w. complexes with IgG has been shown to significantly inhibit the growth of Meth A fibrosarcomas in BALB/c mice. Although SpA reportedly is a potent T cell mitogen that can induce immune cell proliferation and production of humoral factors with anti-tumor activity, it has been suggested that mitogenic enterotoxin contaminants might be responsible for these effects. The purpose of the present study was to investigate the nature of SpA-induced cell proliferation and the relationship between mitogenicity and the anti-tumor effect that we observed in our mouse model. SpA stimulated the proliferation of a mixed population of splenic B and T cells from BALB/c mice, but activity did not require the presence of IgG in the culture medium. Furthermore, mitogenic activity could be inhibited completely by anti-SEA plus anti-SEB, but was unaffected by anti-SpA. HPLC-purified SpA was inactive while the mitogenic factor(s) had the same retention time as authentic enterotoxin and its activity was inhibited by anti-SEA and anti-SEB, but not by anti-SpA. Enterotoxin-free rSpA produced in Escherichia coli had the same IgG binding capacity as the staphylococcal product but was not mitogenic. These data indicate that SEA and SEB completely account for mitogenicity in SpA preparations. In contrast, we found that optimal concentrations of rSpA as well as crude and HPLC purified staphylococcal SpA were equally effective in inhibiting the growth of established Meth A fibrosarcomas demonstrating that SpA is responsible for antitumor activity without any apparent role for enterotoxins.     

Identification of novel human aggrecan T cell epitopes in HLA-B27 transgenic mice associated with spondyloarthropathy

The pathology of ankylosing spondylitis, reactive arthritis, and other spondyloarthropathies (SpA) is closely associated with the human leukocyte class I Ag HLA-B27. A characteristic finding in SpA is inflammation of cartilage structures of the joint, in particular at the site of ligament/tendon and bone junction (enthesitis). In this study, we investigated the role of CD8+ T cells in response to the cartilage proteoglycan aggrecan as a potential candidate autoantigen in BALB/c-B27 transgenic mice. We identified four new HLA-B27-restricted nonamer peptides, one of them (no. 67) with a particularly strong T cell immunogenicity. Peptide no. 67 immunization was capable of stimulating HLA-B27-restricted, CD8+ T cells in BALB/c-B27 transgenic animals, but not in wild-type BALB/c mice. The peptide was specifically recognized on P815-B27 transfectants by HLA-B27-restricted CTLs, which were also detectable by HLA tetramer staining ex vivo as well as in situ. Most importantly, analysis of the joints from peptide no. 67-immunized mice induced typical histological signs of SpA. Our data indicate that HLA-B27-restricted epitopes derived from human aggrecan are involved in the induction of inflammation (tenosynovitis), underlining the importance of HLA-B27 in the pathogenesis of SpA.     

Bacterial cell wall-expressed protein A triggers supraclonal B-cell responses upon in vivo infection with Staphylococcus aureus

We have previously shown that staphylococcal protein A (SpA) anchored to the cell wall of Staphylococcus aureus acts as a virulence factor in septic arthritis. Apart from the ability of SpA to interact with Fcgamma, it also binds to Fab-regions with immunoglobulin heavy chains encoded by the V(H) clan III gene family. The objective of the present study was to investigate whether in vivo expression of SpA by staphylococci induces V(H)III-dependent supraclonal B-cell responses, and whether such responses may affect the ability of the host to produce anti-staphylococcal antibodies. Upon primary infection of mice, a SpA-expressing staphylococcal strain gave rise to significantly higher serum levels of V(H)III-encoded antibodies specific for SpA devoid of Fcgamma-binding ability (MSpA) than an isogeneic spa deletion mutant strain. The V(H)III-dependence of MSpA-specific antibody responses was affected by the size of the staphylococcal inoculum, and differed for IgM and IgG isotypes. Mice that had recovered from a prior mild infection from a SpA-expressing strain were protected against infection-induced weight loss upon reinfection. Although no lasting MSpA-specific IgG was induced by previous mild infection, these protected mice possessed IgG specific for clumping factor A, a conventional staphylococcal protein antigen. Our findings demonstrate that the expression of a B-cell superantigen during staphylococcal infection causes supraclonal changes to the immune system. Notably, while superantigen-triggered B-cell responses do not favor the development of SpA-specific memory B-cells, such responses do not interfere with the development of antibodies specific for a staphylococcal protein antigen associated with protective immunity.     

Staphylococcal protein a deletes B-1a and marginal zone B lymphocytes expressing human immunoglobulins: an immune evasion mechanism

Protein A (SpA) of Staphylococcus aureus is endowed with the capacity to interact with the H chain variable region (V(H)) of human Abs and to target >40% of B lymphocytes. To investigate whether this property represents a virulence factor and to determine the in vivo consequences of the confrontation of SpA with B lymphocytes, we used transgenic mice expressing fully human Abs. We found that administration of soluble SpA reduces B-1a lymphocytes of the peritoneal cavity and marginal zone B lymphocytes of the spleen, resulting in a markedly deficient type 2 humoral response. Single-cell PCR analysis and sequencing of the Ab V(H) gene repertoire revealed a significant reduction of V(H)3+ marginal zone B cells. Since the two B lymphocyte subsets targeted are involved in innate immune functions, our data suggest that crippling of humoral immunity by S. aureus represents an immune evasion mechanism that may aggravate recurrent infections.     

Infiltration of the synovial membrane with macrophage subsets and polymorphonuclear cells reflects global disease activity in spondyloarthropathy

Considering the relation between synovial inflammation and global disease activity in rheumatoid arthritis (RA) and the distinct but heterogeneous histology of spondyloarthropathy (SpA) synovitis, the present study analyzed whether histopathological features of synovium reflect specific phenotypes and/or global disease activity in SpA. Synovial biopsies obtained from 99 SpA and 86 RA patients with active knee synovitis were analyzed for 15 histological and immunohistochemical markers. Correlations with swollen joint count, serum C-reactive protein concentrations, and erythrocyte sedimentation rate were analyzed using classical and multiparameter statistics. SpA synovitis was characterized by higher vascularity and infiltration with CD163⁺ macrophages and polymorphonuclear leukocytes (PMNs) and by lower values for lining-layer hyperplasia, lymphoid aggregates, CD1a⁺ cells, intracellular citrullinated proteins, and MHC–HC gp39 complexes than RA synovitis. Unsupervised clustering of the SpA samples based on synovial features identified two separate clusters that both contained different SpA subtypes but were significantly differentiated by concentration of C-reactive protein and erythrocyte sedimentation rate. Global disease activity in SpA correlated significantly with lining-layer hyperplasia as well as with inflammatory infiltration with macrophages, especially the CD163⁺ subset, and with PMNs. Accordingly, supervised clustering using these synovial parameters identified a cluster of 20 SpA patients with significantly higher disease activity, and this finding was confirmed in an independent SpA cohort. However, multiparameter models based on synovial histopathology were relatively poor predictors of disease activity in individual patients. In conclusion, these data indicate that inflammatory infiltration of the synovium with CD163⁺ macrophages and PMNs as well as lining-layer hyperplasia reflect global disease activity in SpA, independently of the SpA subtype. These data support a prominent role for innate immune cells in SpA synovitis and warrant further evaluation of synovial histopathology as a surrogate marker in early-phase therapeutic trials in SpA.     

Radioactive labels for Protein A: evaluation in the indirect immunoradiometric assay (IRMA) for Bacillus anthracis spores

Staphylococcus aureus Protein A (SpA) labelled with [125I] by the Bolton & Hunter (1973) method performed about as well as labelled sheep anti-rabbit globulin (SAR) in an indirect immunoradiometric assay (IRMA) for Bacillus anthracis spores immobilized on multispot microscope slides. SpA labelled with [3H] by propionylation also performed well but would be expensive to use. SpA labelled with [3H] fluorodinitrobenzene, or labelled with [125I] by the chloramine T reaction gave erratic assay results, high noise values and low signal-to-noise ratios, indicating substantial direct binding of labelled SpA to the slide surface and to the bacterial preparation. The uptake of radioactively labelled SpA in the IRMA was compared with the fluorescence intensity of individual spores in a microfluorometric immunofluorescence (IF) test involving dual labelled fluorescein-[125I]-SpA. The maximum number of SAR molecules bound to the mixture of spores and cell-free antigens in the B. anthracis IRMA was about twice the maximum number of radioactively labelled SpA molecules bound. The SAR:SpA saturation binding ratio on the surface of the spores, however, was approximately the inverse of this. It is concluded that radioactively-labelled SpA is not recommended in preference to anti-species antibody reagents in bacterial IRMA tests but fluorescein-conjugated SpA deserves further consideration for use in microscope-based IF tests for bacterial antigens.     

Genetic variations in immunoglobulin G3 and association with staphylococcal intra-mammary infections in cattle and buffaloes

Animals (n = 152) suffering with mastitis were used to study association between immunoglobulin G3 (IgG3) genotypes and staphylococcal mastitis. Thus, animals (affected and unaffected) were evaluated using PCR-RFLP. Restriction digestion of amplicons of IgG3 using BstYI showed allele A and, genotypes AC, AB and AA predominated in Karan Fries, Sahiwal and Murrah, respectively. HphI digestion revealed allele A and, genotypes AC and AB in higher frequency in animals of first group of all the breeds. Additionally, genotypes associated with mastitic infection showed predominance of AB (BstYI) in unaffected animals of Sahiwal and Murrah; whereas AC and AA were observed in affected group only. Genotype AB (HphI) was prevalent in unaffected and AC in affected animals of Karan Fries and Sahiwal. In Murrah, AC was common in affected and unaffected animal; while AB remained in affected category. Identified genotypes associated with determinants of SpA gene of S. aureus strains revealed the significant outcome. For example AB (BstYI) was found to be correalted with SpA ≤ 7R; whereas with SpA > 7R in Karan Fries. Genotypes AA and AB were more favorably associated with SpA ≤ 7R and AB with the SpA > 7R in Sahiwal cattle. The genotype AB seemed influenced (100%) with SpA > 7R and AC in SpA ≤ 7R in cases of Murrah. Similarly, AA (HphI) in Karan Fries was more likely to be correlated with SpA ≤ 7R, while AC with SpA > 7R. Overall, the molecular analysis revealed that IgG3 gene could be use for selection of animals against mastitis. However, further investigations on IgG3 needed to aid in identify disease- resistant animal.     

Bacterial Expression Systems Based on a Protein A and Protein G Designed for the Production of Immunogens: Applications to Plasmodium falciparum Malaria Antigens

This article describes expression systems based on staphylococcal protein A (SpA) and streptococcal protein G (SpG) which constitute attractive alternatives for the design and production of fusion proteins containing immunogenic structures. A dual expression system that allows the choice between two fusion partners, two synthetic IgG-binding domains (ZZ) of SpA and the serum albumin-binding region BB of SpG, was developed. Genes encoding antigens are expressed in Escherichia coli in parallel as fusions to ZZ and BB and the produced fusion proteins are affinity-purified on human IgG (ZZ fusions) or human serum albumin (BB fusions). The possibility of using ZZ fusions for immunization and the corresponding BB fusions for analysis of the induced immune responses provides a convenient strategy for the generation and analysis of immune responses to selected immunogenic structures. In addition, the cell surface-attaching regions of SpA have been utilized for cell surface display of heterologous antigens on the surface of the Gram-positive bacterium Staphylococcus xylosus. The dual expression system was used to express synthetic gene constructs and genomic gene fragments encoding immunogenic structures from blood-stage antigens of the malaria parasite Plasmodium falciparum. The fusion proteins produced were highly immunogenic in rabbits, mice, and monkeys and induced antibody and T-cell responses to the expressed antigens. Different applications of the SpA- and SpG-based expression systems are described and the immunological properties of the bacterial fusion partners SpA, ZZ, and BB are discussed.     

A very sensitive enzyme-linked immunoabsorbent assay to Staphylococcal protein A in the presence of immunoglobulins

The production of recombinant hepatitis B virus surface antigen (rHBsAg) purified by immunoaffinity chromatography with monoclonal antibodies is used to obtain a vaccine against this virus. Monoclonal antibodies to rHBsAg from mouse ascites have been purified by Staphylococcal Protein A (SpA)--prior coupling to Sepharose CL-4B (Amersham-Bioscences, Uppsala, Sweden). A high sensitivity immunoassay has been developed for the quantification of part-per-million of SpA contaminants likely to co-purify with monoclonal antibodies obtained by Protein A affinity chromatography, in the presence of immunoglobulins. Specific sheep polyclonal Abs against SpA (SpAc1) were used as plate coating and the SpA detection was possible thanks to the conjugates of sheep Ab fragments F(ab)(2) (fSpAc1) and horseradish peroxidase (fSpAc1-peroxidase), reducing the possible unspecific interaction between SpA and Fc fragments. The immunoassay was shown to be specific for SpA contaminants. The quantification limit of the assay was 0.39 ng/ml spreading to the measurement of contamination levels less than 2 ppm of SpA in final preparations of monoclonal antibodies used for the immunopurification of pharmaceutical products, which is quite low for this application.     

Decreased plasma levels of soluble CD18 link leukocyte infiltration with disease activity in spondyloarthritis

Introduction

Spondyloarthritis (SpA) comprises a group of diseases often associated with HLA-B27 and characterized by inflammation of the entheses and joints of the axial skeleton. The inflammatory process in SpA is presumably driven by innate immune cells but is still poorly understood. Thus, new tools for monitoring and treating inflammation are needed. The family of CD18 integrins is pivotal in guiding leukocytes to sites of inflammation, and CD18 hypomorphic mice develop a disease resembling SpA. Previously, we demonstrated that altered soluble CD18 (sCD18) complexes in the blood and synovial fluid of patients with arthritis have anti-inflammatory functions. Here, we study the mechanisms for these alterations and their association with SpA disease activity.

Methods

Plasma levels of sCD18 in a study population with 84 patients with SpA and matched healthy controls were analyzed with a time-resolved immunoflourometric assay (TRIFMA). Binding of sCD18 to endothelial cells and fibroblast-like synoviocytes (FLSs) was studied with confocal microscopy. Shedding of CD18 from peripheral blood mononuclear cells (PBMCs) was studied with flow cytometry and TRIFMA.

Results

Plasma levels of sCD18 were decreased in patients with SpA compared with healthy volunteers (P <0.001), and the lowest levels were in the HLA-B27-positive subgroup (P <0.05). In a multiple regression model, the sCD18 levels exhibited an inverse correlation with the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) (P <0.05), the level of morning stiffness (P <0.05), the Bath Ankylosing Spondilitis Metrology Index (P <0.05), the physician global assessment score (P <0.01), and the sacroiliac magnetic resonance imaging activity score (P <0.05). The mechanisms for these changes could be simulated in vitro. First, sCD18 in plasma adhered to inflammation-induced intercellular adhesion molecule 1 (ICAM-1) on endothelial cells and FLS, indicating increased consumption. Second, CD18 shedding from SpA PBMCs correlated inversely with the BASDAI (P <0.05), suggesting insufficient generation. CD18 was shed primarily from intermediate CD14⁺⁺ CD16⁺ monocytes, supporting the view that alterations in innate immunity can regulate the inflammatory processes in SpA.

Conclusions

Taken together, the failure of patients with SpA to maintain adequate sCD18 levels may reflect insufficient CD18 shedding from monocytes to counterbalance the capture of sCD18 complexes to inflammation-induced ICAM-1. This could increase the availability of ICAM-1 molecules on the endothelium and in the synovium, facilitating leukocyte migration to the entheses and joints and aggregating disease activity.     

Survival of TNF antagonists in spondylarthritis is better than in rheumatoid arthritis. Data from the Spanish registry BIOBADASER

The aim of the present work is to compare drug survival and safety of infliximab, etanercept, and adalimumab (tumor necrosis factor [TNF] antagonists) in spondylarthritis (SpA) with those of rheumatoid arthritis (RA). To this purpose, we analysed the data in BIOBADASER (2000–2005), a drug registry launched in 2000 for long-term follow-up of the safety of these biologics in rheumatic diseases. The rates of drug discontinuation and adverse events (AEs) in SpA (n = 1,524) were estimated and compared with those of RA (n = 4,006). Cox regression analyses were used to adjust for independent factors. Total exposure to TNF antagonists for SpA was 2,430 patient-years and 7,865 for RA. Drug survival in SpA was significantly greater than in RA at 1, 2, and 3 years. The hazard ratio (HR) for discontinuation in SpA compared with RA was 0.66 (95% confidence interval [CI], 0.57–0.76) after adjustment for age, gender, and use of infliximab. The difference remained after controlling for the individual medication and its place in the sequence of treatment. There were fewer SpA patients with AEs (17%) than RA patients (26%; p < 0.001). The HR for AEs in SpA was 0.80 (95% CI, 0.70–0.91) compared with RA after adjustment for age, disease duration, and use of infliximab. In conclusion, due in part to a better safety profile, survival of TNF antagonists in SpA is better than in RA. TNF antagonists are at present a safe and effective therapeutic option for long-term treatment of patients with SpA failing to respond to traditional drugs. Because chronic therapy is necessary, continual review of this issue is necessary.     

Radioactive labels for Protein A: evaluation in the indirect immunoradiometric assay (IRMA) for Bacillus anthracis spores

P hillips , A.P. & M artin , K.L. 1984. Radioactive labels for Protein A: evaluation in the indirect immunoradiometric assay (IRMA) for Bacillus anthracis spores. Journal of Applied Bacteriology 56 , 449–456.
Staphylococcus aureus Protein A (SpA) labelled with [¹²⁵I] by the Bolton & Hunter (1973) method performed about as well as labelled sheep anti-rabbit globulin (SAR) in an indirect immunoradiometric assay (IRMA) for Bacillus anthracis spores immobilized on multispot microscope slides. SpA labelled with [³H] by propi-onylation also performed well but would be expensive to use. SpA labelled with [³H] fluorodinitrobenzene, or labelled with [¹²⁵I] by the chloramine T reaction gave erratic assay results, high noise values and low signal-to-noise ratios, indicating substantial direct binding of labelled SpA to the slide surface and to the bacterial preparation. The uptake of radioactively labelled SpA in the IRMA was compared with the fluorescence intensity of individual spores in a micro-fluorometric immunofluorescence (IF) test involving dual labelled fluorescein-[¹²⁵I]-SpA. The maximum number of SAR molecules bound to the mixture of spores and cell-free antigens in the B. anthracis IRMA was about twice the maximum number of radioactively labelled SpA molecules bound. The SAR : SpA saturation binding ratio on the surface of the spores, however, was approximately the inverse of this. It is concluded that radioactively-labelled SpA is not recommended in preference to anti-species antibody reagents in bacterial IRMA tests but fiuorescein-conjugated SpA deserves further consideration for use in microscope-based IF tests for bacterial antigens.     

Mitogenic response of human and murine T lymphocytes to staphylococcal protein A: Not mediated by binding to cell surface immunoglobulins

Staphylococcal protein A (SpA) stimulates human lymphocytes more efficiently than murine lymphocytes. The subsets of lymphocytes responding to SpA were heterogeneous. In the subsets, cortisone resistant, low Thy 1,2, minor populations of murine thymocytes and mature peripheral T lymphocytes were predominantly stimulated by SpA. Though SpA stimulated cortisone-resistant thymocytes, further treatment of these cells with anti-mouse immunoglobulin sera and guinea pig complement failed to influence the mitogenic response to SpA. It seems likely that SpA contains a substance stimulating T lymphocytes, through binding to sites other than the surface immunoglobulins.     

Staphylococcus aureus protein A activates TNFR1 signaling through conserved IgG binding domains

Staphylococcus aureus continues to be a major cause of infection in normal as well as immunocompromised hosts, and the increasing prevalence of highly virulent community-acquired methicillin-resistant strains is a public health concern. A highly expressed surface component of S. aureus, protein A (SpA), contributes to its success as a pathogen by both activating inflammation and by interfering with immune clearance. SpA is known to bind to IgG Fc, which impedes phagocytosis. SpA is also a potent activator of tumor necrosis factor alpha (TNF-alpha) receptor 1 (TNFR1) signaling, inducing both chemokine expression and TNF-converting enzyme-dependent soluble TNFR1 (sTNFR1) shedding, which has anti-inflammatory consequences, particularly in the lung. Using a collection of glutathione S-transferase fusions to the intact IgG binding region of SpA and to each of the individual binding domains, we found that the SpA IgG binding domains also mediate binding to human airway cells. TNFR1-dependent CXCL8 production could be elicited by any one of the individual SpA IgG binding domains as efficiently as by either the entire SpA or the intact IgG binding region. SpA induction of sTNFR1 shedding required the entire IgG binding region and tolerated fewer substitutions in residues known to interact with IgG. Each of the repeated domains of the IgG binding domain can affect multiple immune responses independently, activating inflammation through TNFR1 and thwarting opsonization by trapping IgG Fc domains, while the intact IgG binding region can limit further signaling through sTNFR1 shedding.     

Partitioning of beta-galactosidase fusion proteins in PEG/potassium phosphate aqueous two-phase systems.

Four different beta-galactosidase fusion proteins have been partitioned in poly(ethylene glycol) (PEG) 4000/potassium phosphate aqueous two-phase systems. The partition coefficients (K) of staphylococcal protein A-beta-galactosidase (SpA beta gal) (K = 3.5) and staphylococcal protein A-streptococcal protein G-beta-galactosidase (AG beta gal) (K = 2.8) were compared with the partition coefficients of their constituent molecules, beta-galactosidase, SpA, and protein AG. It was found that by fusing beta-galactosidase to the smaller proteins SpA and protein AG, their partition coefficients were increased four to five times. Experimental data were fitted into, and found to agree with, the Albertsson partition model of interacting molecules. The compatibility with PEG and potassium phosphate of beta-galactosidase, SpA, and two different versions of the SpA beta gal protein, displayed as precipitation curves, showed a relationship to the protein partition coefficients in PEG/potassium phosphate systems. High solubility in one phase component was accompanied by preferential partitioning to the phase rich in the same component in the PEG/potassium phosphate system. Also, a changed linker region in SpA beta gal resulted in a more soluble protein. This, together with the improved K values of the target proteins by fusion, shows that it is possible to use beta-galactosidase as an affinity handle.     

Electron microscopic and hydrodynamic studies of protein A-immunoglobulin G soluble complexes

The soluble complexes formed by reacting staphylococcal protein A (SpA) with rabbit immunoglobulin G (IgG) antibodies were characterized by hydrodynamic and electron microscopic methods. In moderate SpA excess, equilibrium mixtures of SpA and rabbit IgG formed four discrete complexes that sedimented at approximately 7, 10, 13, and 15S. The putative complexes were visible by electron microscopy and appeared to contain one, two, three, and approximately five molecules of IgG. Probably because of its elongated shape, SpA was not clearly visible in these mixtures or in control preparations of SpA alone. Both native IgG and IgG modified by cleavage of its single-hinge disulfide bond formed similar complexes on interaction with SpA. It was possible to resolve heterogeneous mixtures of IgG-SpA complexes by using an analytical ultracentrifuge equipped with a photoelectric scanner interfaced to a small computer. The relative concentrations and sedimentation velocities of different complexes in a mixture were determined from computer-generated integral and derivative plots. Both hydrodynamic and electron microscopic methods revealed that the distribution of complexes was sensitive to the IgG to SpA molar ratio. The relative amounts of faster complexes increased as the IgG to SpA molar ratio was increased. Surprisingly, when the IgG to SpA molar ratio was greater than or equal to 2, the complexes were converted into a unique 17S complex. This rather unprecedented transformation was reversible: the addition of excess SpA caused the dissociation of the 17S complex into a mixture of the 7, 10, 13, and 15S structures. The average translational diffusion coefficient of the 17S complex was 2.62 +/- 0.13 Ficks. In the electron microscope, the complex appeared to be exceptionally compact with an average diameter of 287 A. The stoichiometry of the 17S complex, together with sedimentation equilibrium, diffusion, and electron microscopic measurements, indicated that it is composed of four molecules of IgG and two molecules of SpA.     

Triangular Relationship between Sleep Spindle Activity,General Cognitive Ability and the Efficiency of Declarative Learning

EEG sleep spindle activity (SpA) during non-rapid eye movement (NREM) sleep has been reported to be associated with measures of intelligence and overnight performance improvements. The reticular nucleus of the thalamus is generating sleep spindles in interaction with thalamocortical connections. The same system enables efficient encoding and processing during wakefulness. Thus, we examined if the triangular relationship between SpA, measures of intelligence and declarative learning reflect the efficiency of the thalamocortical system. As expected, SpA was associated with general cognitive ability, e.g. information processing speed. SpA was also associated with learning efficiency, however, not with overnight performance improvement in a declarative memory task. SpA might therefore reflect the efficiency of the thalamocortical network and can be seen as a marker for learning during encoding in wakefulness, i.e. learning efficiency.     

Staphylococcus aureus protein A induced inflammatory response in human corneal epithelial cells

In the present study, we examined the role of Staphylococcus aureus protein A (SpA) in inducing inflammatory response in human corneal epithelial cells (HCECs). Exposure of HCECs to SpA induces rapid NF-kappaB activation and secretion of proinflammatory cytokine/chemokines (TNF-alpha and IL-8) in both concentration and time-dependent manner. Challenge of HCECs with live SpA(-/-) mutant S. aureus strains resulted in significantly reduced production of the cytokines when compared to the wild-type S. aureus strain. SpA also elicited the activation of MAP Kinases P38, ERK, but not JNK, in HCECs. SpA-induced production of proinflammatory cytokine were completely blocked by the NF-kappaB and p38 inhibitors and partially inhibited by the Jnk inhibitor. Pretreatment with anti-TLR2 neutralizing antibody had no effect on SpA-induced inflammatory response in HCECs, suggesting that this response is independent of TLR2 signaling. Moreover, unlike TLR2 ligands, SpA failed to induce the expression of antimicrobial peptides (hBD2 and LL-37) in HCECs. These studies indicate that SpA is a S. aureus virulence factor that stimulates HCEC inflammatory response through a pathway distinct from TLR2 in HCECs.     

Adoptive transfer of a superantigen-induced hole in the repertoire of natural IgM-secreting cells.

We have recently evaluated the host response to the bacterial toxin, protein A from Staphylococcus aureus (SpA), which has the capacity to interact with B-cell antigen receptors encoded by V(H) clan III genes via a conserved variable region framework surface. In these studies, intraperitoneal instillation of SpA induced a persistent T-cell independent loss of a large supraclonal set of susceptible lymphocytes, which includes clan III/V(H) S107 family-expressing B-1 cells and their antibody products. To determine whether these long-term effects could represent the influence of residual in vivo deposited superantigen, we have now performed adoptive transfer of peritoneal B cells from superantigen- and control-treated donors. These studies demonstrated that mice that received cells from SpA-treated donors also exhibited the same induced supraclonal hole in the expressed repertoire of natural IgM-secreting cells due to supraclonal deletion. These studies clarify the cellular mechanisms responsible for B-cell superantigen-induced modification of the repertoires of in vivo polyclonal B-cell populations.     

Chronically inflamed synovium from spondyloarthropathy and rheumatoid arthritis investigated by protein expression profiling followed by tandem mass spectrometry

We investigated the cytosolic proteome of inflamed synovial tissue by hierarchical clustering analysis and validated the feasibility of this proteome analysis by identifying proteins that were differentially expressed between rheumatoid arthritis (RA), spondyloarthropathy (SpA), and osteoarthritis (OA). Synovial biopsy samples were obtained from 18 patients undergoing needle arthroscopy for knee synovitis associated with RA (n = 6) and SpA (n = 6), and for joint effusion of the knee associated with OA (n = 6). Cytosolic proteins were extracted from the tissue and subjected to two-dimensional gel electrophoresis. Protein expression patterns were statistically analyzed and used for hierarchical cluster analysis. Proteins of interest were independently identified by matrix-assisted laser desorption/ionization- and electrospray ionization-mass spectrometry. Hierarchical cluster analysis of the complete match set, containing 640 spots, remarkably segregated SpA from RA and OA. Next, we used a subset of spots that was statistically, differentially expressed (P < 0.01), between RA and SpA, SpA and OA, or RA and OA, in both Student's t-test and Mann-Whitney U-test. The dendrograms revealed distinct clustering of RA versus SpA and RA versus OA. Spots that were differentially expressed between the groups were identified by tandem mass spectrometry. Fructose bisphosphate aldolase A and alpha-enolase showed higher expression levels in SpA than in OA (P < 0.01). Calgranulin A myeloid related protein-8 (MRP-8) was markedly up-regulated in RA and SpA patients in comparison to OA patients where this spot was below detection limit. The analysis of the cytosolic proteome of synovial tissue is a useful approach to identify disease-associated proteins in chronic inflammatory arthritis.