Tumoricidal adsorption of Staphylococcus aureus organisms on Ehrlich ascites tumor cells sensitized with rabbit antibody

A tumoricidal effect was observed when protein A-bearing Staphylococcus aureus organisms were adsorbed on Ehrlich ascites tumor cells previously sensitized with antiserum from a rabbit immunized with Ehrlich ascites tumor cells. Electron micrographs showed that staphylococci were firmly attached to the tumor cells, which might explain how effectively the attached cocci killed the tumor cells. The tumoricidal effect was confirmed not only by an in vitro experiment but also by an in vivo one. The possible applications of the tumoricidal adsorption as an indicator for staphylococcal virulence or for selective anti-tumor action was discussed.     

Immunoglobulin M synthesized by human lymphoblastoid cells: interaction with Staphylococcus aureus and protein A.

Immunoglobulin M synthesized by a human lymphoblastoid cell line, LA173, was found to bind specifically to the protein A-bearing Cowan I strain of Staphylococcus aureus. The (3H)-leucine-labeled, secreted IgM from these LA173 cells also formed precipitin complexes with purified protein A. Soluble complexes formed at high protein A/IgM ratios retained the ability to bind to the bacterial surface. Precipitin complexes also were observed in double diffusion Ouchterlony gels with a line of identity formed between the IgM, protein A, and anti-IgM in adjacent wells. Intracellular IgM species from detergent-lysed LA173 cells were bound to S. aureus. Labeled 19S pentamers, 8S monomers, and HL subunits were eluted from the bacteria and identified by velocity sedimentation and SDS agarose-acrylamide gel electrophoresis. In addition, several intermediates migrating between 8S and 19S were detected and shown to contain authentic H and L chains. Binding of the labeled IgM 19S pentamers to staphylococci was not inhibited by prior treatment of the bacteria with an excess of unlabeled human IgG. However, S. aureus saturated with unlabeled IgG did not bind either labeled IgM monomers or labeled IgG. The interaction of this human IgM with S. aureus exhibited a high degree of specificity with quantitative recovery of secreted 19S IgM. Intracellular IgM species were bound selectively by the bacteria with little if any contamination by other cytoplasmic proteins.     

Staphylococcus aureus protein A triggers T cell-independent B cell proliferation by sensitizing B cells for TLR2 ligands

B cells possess functional characteristics of innate immune cells, as they can present Ag to T cells and can be stimulated with microbial molecules such as TLR ligands. Because crude preparations of Staphylococcus aureus are frequently used as polyclonal B cell activators and contain potent TLR2 activity, the scope of this study was to analyze the impact of S. aureus-derived TLR2-active substances on human B cell activation. Peripheral B cells stimulated with chemically modified S. aureus cell wall preparations proliferated in response to stimulation with crude cell wall preparations but failed to be activated with pure peptidoglycan, indicating that cell wall molecules other than peptidoglycan are responsible for B cell proliferation. Subsequent analysis revealed that surface protein A (SpA), similar to BCR cross-linking with anti-human Ig, sensitizes B cells for the recognition of cell wall-associated TLR2-active lipopeptides (LP). In marked contrast to TLR7- and TLR9-triggered B cell stimulation, stimulation with TLR2-active LP and SpA or with crude cell wall preparations failed to induce IgM secretion, thereby revealing qualitative differences in TLR2 signaling compared with TLR7/9 signaling. Notably, combined stimulation with SpA plus TLR2 ligands induced vigorous proliferation of a defined B cell subset that expressed intracellular IgM in the presence of IL-2. Conclusion: S. aureus triggers B cell activation via SpA-induced sensitization of B cells for TLR2-active LP. Combined SpA and TLR2-mediated B cell activation promotes B cell proliferation but fails to induce polyclonal IgM secretion as seen after TLR7 and TLR9 ligation.     

Solid phase radioimmunoassay for quantitation of antigen-specific IgG in human sera with 125I-protein A from Staphylococcus aureus.

Radiolabeled protein A from Staphylococcus aureus (Staph A) has been used to develop a solid phase, noncompetitive radioimmunoassay for quantitation of specific IgG antibody. The assay involves two incubations: First, agarose-insolubilized antigen is mixed with serum samples for 1 to 4 hr during which specific antibody is bound; second, after a washing procedure, the solid phase immune complexes are incubated for 4 to 18 hr with 125I-Staph A, during which the radiolabeled detection protein binds to the insolubilized specific IgG antibody. In a comparative study of the IgG antiphospholipase A antibody content of 23 human sera drawn from honeybee venom-sensitive patients, resulted of the Staph A assay correlated highly (r = 0.981, p less than 0.001, N = 23) with those obtained from a liquid phase, competitive radioimmunoprecipitation (double antibody) assay. The two assays demonstrated comparable precision, sensitivity, and reproducibility. In contrast, the use of 125I-Staph A in the solid phase radioimmunoassay was superior to 125I rabbit anti-human IgG because of lower negative serum (blank) values, shorter time required to reach equilibrium binding, and greater precision and reproducibility. In principle, the 125I Staph A assay may be applied ot IgG quantitation for crude allergen extracts as well as purified antigens. Furthermore, the sera of a number of mammalian species may be studied without further modification.     

Protein A research in 1200 strains of Staphylococcus (using a rapid detection technic by passive hemagglutination with glutaraldehyde treated erythrocytes]

The technique used was that of passive hemaglutination of red cells of sheep prepared with glutaraldehyde and sensitized by anti-erythrocyte antibodies. A protein-A was not found in S. epidermidis strains, but was present in 96.3 per cent of 689 strains of S. aureus from human, animal and food origins.     

Induction of monocytic suppression after stimulation of peripheral human mononuclear cells with staphylococcal protein A and Staphylococcus aureus

Staphylococcal protein-A (SPA) and Staphylococcus aureus are known to be polyclonal human B-cell activators. It was noted that they induced plaque-forming-cell (PFC) responses lower than those induced by pokeweed mitogen (PWM) and the possibility of early triggering of a suppressor cell was investigated in the present series of experiments. Peripheral mononuclear cells (MNC) were passed through Sephadex G-10 columns to eliminate monocytes. The PFC responses to SPA and S. aureus were thereby increased. PWM-driven PFC responses are suppressed by the simultaneous presence of SPA in a dose-related way, if present in the early phases of the cultures. MNC precultured with SPA or S. aureus have the ability to suppress the PFC response of autologous MNC to PWM. Interestingly this suppressor cell activity was radiation resistant and could not be abrogated by treatment with anti-T-cell monoclonal antibody plus complement. The above experiments clearly demonstrate that the observed low PFC responses of MNC after stimulation with SPA and S. aureus are due to the induction of suppressor cells by these stimulants. The suppressor cells are apparently of monocytic origin.     

New method for typing Staphylococcus aureus resistant to methicillin based on sulphur-35 methionine labelled proteins: its application in an outbreak.

Sodium dodecyl sulphate polyacrylamide gel electrophoresis of sulphur-35 methionine labelled cellular proteins of Staphylococcus aureus resistant to methicillin was used as a typing method during an outbreak on a cardiothoracic ward. This showed that the outbreak strain was indistinguishable from the epidemic strain of methicillin resistant S aureus prevalent in London. In contrast, 44 epidemiologically separate strains of S aureus gave individually distinct radiolabelled protein profiles. This method permitted rapid confirmation that an epidemic strain was responsible and indicated the need for urgent control measures.     

Human T lymphocyte receptor for sheep erythrocytes: Characterization of a specific antiserum and its application in the detection and quantitation of the receptor in a soluble form

An anti-human T lymphocyte serum specific to the receptor for sheep erythrocytes (E) was produced by immunizing sheep with the complex autologous E-soluble E receptor (ER_s). The soluble receptor (R_s) was obtained by heating human lymphocytes at 45 °C for 1 hr. The anti-R_s serum has been shown to inhibit E-rosette formation, to be cytotoxic to T cells, to identify T lymphocytes by indirect immunofluorescence, and to stimulate blastogenesis. The reaction of anti-R_s with R_s was directly demonstrated by two newly developed methods: agglutination of complexes formed by the treatment of formolized E with R_s (EFR_s complexes) and adhesion of a protein A producer strain of Staphylococcus aureus to EFR_s treated with anti-R_s. The anti-R_s antibodies could be neutralized by R_s present in supernatant of heated peripheral lymphocytes, inhibiting the above reactions and therefore providing methods to quantitate R_s in biological preparations. The importance of these assays is that R_s plays an immunoregulatory activity, and high levels of R_s in serum are associated with depressed cell-mediated immunity.     

A simple purification method for enterotoxin F produced by Staphylococcus aureus and some properties of the toxin

Staphyloccoccus aureus enterotoxin F (SEF), which is associated with S. aureus strains isolated from toxic-shock-syndrome patients, was purified by successive chromatography on CM sephadex C-25 and gelfiltration on sephadex G-75. When tested by disc-polyacrylamide gel-electrophoresis the toxin migrated as a homogeneous protein. In SDS-polyacrylamide gel-electrophoresis three protein bands were observed. The main component had a mol wt of 23000 and the two minor components had a mol wt<13 000. By iso-electric focussing a main protein band with an iso-electric point of 7.2 was obtained. The LD₅₀ for rabbits (3–3.5 kg) by subcutaneous and intravenous application of SEF was 6 g and 180 g, respectively. Antibodies to SEF prepared in a sheep did not react with other staphylococcal enterotoxins (A to E).     

A new feasible method for fibrinogen purification based on the affinity of Staphylococcus aureus clumping factor A to fibrinogen

Plasma fibrinogen participates in several physiological and pathological events thus becoming a useful studying material in biomedical research. Here we report a new convenient method for fibrinogen purification based on the affinity of Staphylococcus aureus clumping factor A to fibrinogen. Clumping factor A (ClfA) is a cell wall-anchored surface protein of S. aureus bacteria that binds with a high affinity to the fibrinogen gamma chain C-terminus via a segment encompassing the residues 221-550. This activity of ClfA (ClfA(221-550)) was produced in fusion to the C-terminus of glutathione-S-transferase (GST) with recombinant technology and used as an affinity ligand to capture plasma fibrinogen. GST-ClfA(221-550) fusion protein was immobilized onto the glutathione-conjugated beads packed in a plastic column by its GST part. Then, this affinity column was loaded with citrated and heparinized human plasma. After washing out unbound proteins, column-captured fibrinogen was specifically eluted down with a citrate buffer solution (50mM, pH 5.6). Purified human fibrinogen exhibited the ability to support platelet adhesion and aggregation and formed fibrin clot by thrombin, indicating that ClfA(221-550)-purified human fibrinogen is a functionally active product. We also found that both the rat and mouse fibrinogens could be purified as well as human fibrinogen with this method. By virtue of its simplicity and feasibility, ClfA(221-550)-based method would be very useful to the investigators who need fibrinogen to perform their studies.     

Response of human B cells to Staphylococcus aureus Cowan I: T-independent proliferation and T-dependent differentiation to immunoglobulin secretion involve subsets separable by rosetting with mouse erythrocytes

We separated T-depleted mononuclear cells into subsets by rosetting with mouse erythrocytes and studied proliferation and differentiation responses to Staphylococcus aureus Cowan I (SAC), pokeweed mitogen (PWM), and a combination of the two polyclonal activators. All of the T cell-independent proliferation of unfractionated B cells in response to SAC was attributable to mouse erythrocyte rosette-forming cells (BMR+). BMR- cells were not stimulated to proliferate by SAC in the presence or absence of T cells, but did proliferate to PWM plus irradiated T cells. Co-stimulation of BMR+ cells with SAC and PWM in the presence of autologous T cells did not lead to immunoglobulin secretion. The B cells stimulated to divide by SAC apparently do not become responsive to B cell differentiation factors and are distinct from those that undergo T cell-dependent differentiation.