Dynamics of staphylococcal infection in guinea pigs with delayed hypersensitivity to Staphylococcus aureus surface antigens

The relationship between delayed hypersensitivity (DH) to S. aureus surface antigens and the intensity of the infectious process induced by the sublethal infection of guinea pigs with S. aureus was studied. The protective effect, manifested by a decrease in the staphylococcal contamination of the spleen tissue and by an increase in the level of the activation of lymphocytes, was shown to correlate with DH induced by inactivated staphylococcal cells. In infected guinea pigs having DH to different staphylococcal antigens the disease either took a more severe course (in cases of DH to cell wall or peptidoglycan) than in the animals subjected only to infection, or no aggravation of the disease was observed (in cases of DH to protein A).     

Nematode infections are risk factors for staphylococcal infection in children

Nematode infection may be a risk factor for pyogenic liver abscess in children and we hypothesized that the immunomodulation induced by those parasites would be a risk factor for any staphylococcal infection in children. The present study was designed to compare, within the same hospital, the frequency of intestinal nematodes and Toxocara infection in children with and without staphylococcal infections. From October 1997 to February 1998, 80 children with staphylococcal infection and 110 children with other diseases were submitted to fecal examination, serology for Toxocara sp., evaluation of plasma immunoglobulin levels, and eosinophil counts. Mean age, gender distribution, birthplace, and socioeconomic conditions did not differ significantly between the two groups. Frequency of intestinal nematodes and positive serology for Toxocara, were remarkably higher in children with staphylococcal infections than in the non-staphylococcal group. There was a significant correlation between intestinal nematodes or Toxocara infection and staphylococcal infection in children, reinforced by higher eosinophil counts and higher IgE levels in these children than in the control group. One possible explanation for this association would be the enhancement of bacterial infection by the immunomodulation induced by helminth infections, due to strong activation of the Th2 subset of lymphocytes by antigens from larvae and adult worms.     

The immunomodulating action of the surface antigens of the influenza A virus in experimental staphylococcal infection

The results of the study of influenza A virus surface antigens, hemagglutinin and neuraminidase, in the induction of nonspecific immunomodulation and protection from acute pulmonary staphylococcal infection have been studied. Protective effect, the cell composition of bronchoalveolar lavage fluid depend on the serological subtypes of surface antigens used for intranasal immunization and the infective dose of staphylococci.     

Sensitizing and protective activity of a staphylococcal vaccine made of water-soluble antigens, depending on the method and schedule of administration

An experiment on guinea pigs immunized with staphylococcal vaccine prepared from water-soluble antigens revealed that the degree of developing sensitization and specific resistance was essentially determined by the method and schedule of the administration of the preparation. The intranasal administration of the vaccine induced a lesser degree of sensitization in comparison with its subcutaneous injection. The optimum response to the administration of the vaccine (a low sensitization level and a high degree of protection from infection) was observed in the animals immunized first intranasally and then by subcutaneous injection. The subcutaneous injection of the preparation in combination with its subsequent intranasal application induced a more pronounced degree of sensitization and a lesser degree of protection from infection.     

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.     

Technique for the detection of Toxoplasma gondii antigens in mouse urine

A simple and rapid staphylococcal coagglutination test for the detection of Toxoplasma gondii antigens in mice urine is described. A suspension of protein-A containing Staphylococcus aureus coated with rabbit hyperimmune serum was used as reagent. The sensitivity of the antigen assay was found to be at least 118 ng of the antigen protein per ml. No coagglutination was observed when the reagent was challenged against antigenic solutions of other parasites. The suitability of the method for detecting antigens of T. gondii in urine samples was studied by experimental toxoplasma infection in mice. Before the staphylococcal test, the urine samples were double serially diluted in 0.1 M PBS. From the second day on all samples from infected mice were positive at 1/16 dilution. At this dilution, all samples from non infected mice were negative or did not produce coagglutination. This method might be used in the rapid etiological diagnosis also in human cases of acute toxoplasmosis.     

Effect of trypsin on the immune response in localized staphylococcal infection

The influence of trypsin on the formation of immune response induced by a thymus-dependent antigen at different periods of localized staphylococcal infection has been studied. A single intramuscular injection of bovine trypsin has been found to enhance immune response to sheep red blood cells (SRBC) in healthy mice and, to a still greater degree, in mice with localized staphylococcal infection. the development of localized staphylococcal infection has been shown to have no influence on the manifestation of the immuno-suppressive effect of splenocytes in SRBC-immunized mice or to enhance this effect. The injection of trypsin decreases the immunosuppressive effect of splenocytes in healthy or staphylococcus-infected hyperimmune mice.     

Role of T- and B-lymphocytes in the heterogeneity of human cell-mediated reactions to bacterial antigens]

Leucocytes from 30 patients with allergy to tuberculin and bacterial antigens were treated with antithymus (ATS) and anti-immune globulin (AIGS) sera. The leucocyte migration inhibition test (LMIT) was performed with these antigens. ATS abolished the LMIT induced by tuberculin and sometimes by bacterial antigens (staphylococcal, streptococcal etc.). AIGS frequently abolished the LMIT induced by bacterial antigens, but not by tuberculin. In some cases the treatment with any serum abolished the LMIT induced by the antigens, or, on the contrary, it was abolished only by a successive treatment with both sera. The lymphocyte types (T or B) determining the secondary immune response to the same antigen are different in various patients, as well as they differ in the same patients in relation to diverse antigens. Five types of lymphocyte - antigen interrelation in the LMIT have been distinguished.     

The adhesion of protein A-bearing Staphylococcus aureus organisms to soluble-staphylococcal-antigens-coated HeLa cells mediated by specific antibodies

The adhesion of staphylococcal protein A (SpA)-bearing Staphylococcus aureus Cowan I organisms to HeLa cells was enhanced by pretreatment of HeLa cells with staphylococcal extracellular antigens and antibodies to them. The adhesion of HLj, an SpA-poor mutant derived from Cowan I, to HeLa cells was not enhanced by the same pretreatment of HeLa cells. Furthermore, the enhanced staphylococcal adhesion was inhibited by soluble SpA. The antigen(s) responsible for the enhanced staphylococcal adhesion was(were) heat stable. Pretreatment of HeLa cells with the mixture of staphylococcal extracellular antigens and antibodies to them also enhanced the adhesion of Cowan I. Similarly the adhesion of Cowan I was enhanced by pretreatment of HeLa cells with extracellular antigens of Pseudomonas aeruginosa and antibodies to them. These results indicated that cell-bound SpA mediated the binding of S. aureus to immune complexes composed of extracellular bacterial products and antibodies to them bound to the surface of HeLa cells, and suggested another role of cell-bound SpA as a co-adhesin with other factors in infections due to S. aureus.     

Isolation and characterization of surface antigens from Schistosoma mansoni. II. Antigenicity of radiolabeled proteins from adult worms.

Adult Schistosoma mansoni were radiolabeled in vitro with 125I Bolton-Hunter reagent. Surface membrane antigens were solubilized with non-ionic detergent, then reacted with infection or normal serum. The antigen-antibody complexes were then precipitated with staphylococcal protein A immunoadsorbent, eluted with urea and SDS, and fractionated by SDS-PAGE. The results indicated the presence of 6 to 8 tegument antigens, depending on the type of antisera used. Human antisera to S. japonicum and S. haematobium reacted with some but not all of the antigens identified with human S. mansoni infection serum; this implies the presence of species-specific tegument antigens. The molecular weights of the radiolabeled antigens ranged from 10,000 to 100,000. A large (greater than 100,000) molecular weight glycoprotein and an uncharacterized lipid fraction appeared to be precipitated nonspecifically. Immunoprecipitation methods with anti-mouse IgG and anti-mouse whole serum failed to detect the presence of hostlike antigens in the labeled extracts. Several of the labeled proteins from S. mansoni were found to react with serum from patients infected with either S. haematobium or with S. japonicum.     

Preparation of Staphylococcus aureus antigens for evaluation of their immunological reactivity with the human sera by the western blot method]

Extracellular antigens as well as cell wall extracts of 4 S. aureus strains isolated from different kinds of infection were analysed by Western-Blott technique. Materials obtained in two systems of bacteria cultivation (with and without aeration) were compared. Four systems of PAGE (native conditions, with 8.0 M urea, with SDS and SDS after previous reduction of the material with 2-mercaptoethanol) were compared in order to get the best differentiation of proteins and antigens. Immunological reactivity of the antigens mixture with two human sera: highly positive (with three S. aureus antigens in ELISA) from patient with staphylococcal sepsis and negative (from blood donor) were analysed. The best results were obtained after reduction of the cell wall extracted material in SDS-PAGE. The different protein patterns depending on the strain and the method of bacteria cultivation were observed. The standardisation of Western-Blott technique was performed, including titration of the sera to get the best differentiation of the antigens. The difference in immunological reactivity of the positive and negative sera with staphylococcal antigens mixture showed rather quantitative than qualitative character.     

Protective efficacy of combined administration of the multicomponent vaccine and the immunomodulator myelopid in experimental infections in mice

The influence of myelopid (MP) on the protective activity of polycomponent vaccine VP-4 prepared from the antigens of opportunistic bacteria was studied on experimental infections of mice, caused by Klebsiella pneumoniae, Salmonella typhimurium and Staphylococcus aureus. In staphylococcal and Klebsiella infections the joint administration of vaccine VP-4 and MP produced more pronounced protective effect than each of these preparations, introduced alone. The protective action of vaccine VP-4 was specially enforced by MP in cases of local staphylococcal infection. Recommendations on the joint use of two or more immunomodulating agents are possible only on the basis of the experimental substantiation of their effect in definite infections.     

The role of humoral and cellular mediators in enhanced mammary inflammatory reactions to staphylococcal infection in systemically immunized ewes

Prior systemic immunization with live Staphylococcus aureus vaccine enhances the early recruitment of neutrophils into nonlactating mammary glands infected with staphylococci. The study investigates the role of humoral and cellular mediators in this phenomenon. Intramammary infusion of bacteria suspended in immune sheep serum did not enhance the inflammatory response to infection in nonimmunized ewes despite the presence of complement in the infused serum. Infusion of complement activated by incubation with zymosan evoked a massive neutrophil influx into mammary secretions by 4 hr after infusion. Hemolytic complement activity was not detected in mammary secretions of immunized or nonimmunized ewes. These findings indicate that, despite the inflammatory effect of complement activation, humoral immune factors did not promote neutrophil migration into infected glands. Mammary glands of systemically immunized ewes stimulated 5 days previously with staphylococcal soluble antigens (SSA) supported larger neutrophil influxes during staphylococcal infection than contralateral glands stimulated with endotoxin 5 days prior to infection. Exudates of SSA-stimulated glands had significantly higher cell concentrations, prior to infection, than endotoxin-stimulated glands; however elevated cell concentrations in endotoxin-stimulated glands of nonimmune ewes did not support enhanced inflammatory responses. These findings suggest that qualitative but not quantitative characteristics of mammary leucocytes influence the inflammatory response to infection in systemically immunized ewes.     

Humoral response to Staphylococcus aureus antigens evaluated by the western blotting method]

Cellular antigens extracted from the cells of four Staphylococcus aureus strains from different kinds of infections (sepsis, osteomyelitis, furunculosis) were analysed by the western blotting technique. Antibiotic sensitivity pattern of the strains was compared. One isolate was found to be MRSA strain. Sera samples from patients of whom strains were isolated and four sera from blood donors (as a control) were used in the investigation. IgG levels for purified staphylococcal antigens (lipase, alpha-toxin and teichoic acid) were estimated. Interaction between extracted bacterial antigens and serum antibodies of IgG class were analysed in homologous and heterologous systems. The most strong immunological reaction of the investigated sera with staphylococcal antigens was observed in the case of homologous system. Serum from sepsis patient was found to be the most reactive serum with all staphylococcal antigens mixtures.     

Mitogenic activity of the antigens isolated from different Staphylococcus aureus strains in relation to mouse and guinea pig splenocytes

The mitogenic effect of corpuscular antigens with respect to the splenocytes of animals was found to depend on the strain of Staphylococcus aureus. The maximum synthesis of DNA in the cells was induced by corpuscular antigen Smith and the minimum synthesis, by Wood-46. The synthesis of DNA was activated in both B- and T-splenocytes in response to corpuscular antigens Wood-46, Cowan-1 and Smith, as well as to the cell wall and protein A. Peptidoglycan produced a mitogenic effect only in B-lymphocytes, and teichoic acid showed no mitogenic activity in mouse splenocytes. The mitogenic effect of staphylococcal antigens on splenocytes depended on the dose of the antigen and the time of cultivation. After 48-hour cultivation the incorporation of 3H-thymidine into the DNA of mouse cells was 5 times higher than into the DNA of guinea-pig cells. The optimum mitogenic dose in thymectomized BALB/c mice with respect to splenocytes was higher than in normal BALB/c mice practically by one order.     

Induction of apoptosis by sphingoid long-chain bases in Aspergillus nidulans

Sphingolipid metabolism is implicated to play an important role in apoptosis. Here we show that dihydrosphingosine (DHS) and phytosphingosine (PHS), two major sphingoid bases of fungi, have potent fungicidal activity with remarkably high structural and stereochemical specificity against Aspergillus nidulans. In fact, only naturally occurring DHS and PHS are active. Further analysis revealed that DHS and PHS induce rapid DNA condensation independent of mitosis, large-scale DNA fragmentation, and exposure of phosphatidylserine, all common morphological features characteristic of apoptosis, suggesting that DHS and PHS induce apoptosis in A. nidulans. The finding that DNA fragmentation requires protein synthesis, which implies that an active process is involved, further supports this proposition. The induction of apoptosis by DHS and PHS is associated with the rapid accumulation of reactive oxygen species (ROS). However, ROS are not required for apoptosis induced by DHS and PHS, as scavenging of ROS by a free radical spin trap has no effect. We further demonstrate that apoptosis induced by DHS and PHS is independent of metacaspase function but requires mitochondrial function. Together, the results suggest that DHS and PHS induce a type of apoptosis in A. nidulans most similar to the caspase-independent apoptosis observed in mammalian systems. As A. nidulans is genetically tractable, this organism should be an ideal model system for dissecting sphingolipid signaling in apoptosis and, importantly, for further elucidating the molecular basis of caspase-independent apoptosis.     

Identification of vaccine candidate antigens of Staphylococcus aureus by serological proteome analysis

In this study we applied serological proteome analysis (Klade, C. S. et al. Proteomics 2001, 1, 890-898) for identification of bacterial vaccine candidate antigens. First, approximately one hundred sera from healthy individuals and patients suffering from Staphylococcus aureus infections were screened for antibodies against staphylococcal lysates and recombinant proteins representing surface antigens. Two pools (healthy donors, patients) each consisting of five sera with the highest antiproteinaceous IgG reactivity were selected. Second, S. aureus COL was grown under different conditions and the number of antigens expressed was monitored by Western blot analysis. Third, surface proteins were enriched by digesting the bacterial cell wall under isotonic conditions and subsequent removal of protoplasts. These protein preparations were resolved by two-dimensional electrophoresis (2-DE) (pI 4-7). 2-DE immunoblotting using the preselected serum pools at 1:10 000-1:100 000 dilutions revealed a number of highly immunogenic staphylococcal proteins. Twenty-one spots were isolated by preparative 2-DE, and analysed by matrix-assisted laser desorption/ionization mass spectrometry and tandem mass spectrometry sequencing of tryptic peptides. This led to the identification of 15 proteins including known and novel vaccine candidates. Seroreactivity of several antigens including serine-aspartate repeat containing protein D, immuno-dominant staphylococcal antigen and a novel 309 amino acid lipoprotein was independently confirmed by enzyme-linked immunosorbent assay and Western blot analysis of purified recombinant proteins. In conclusion, serological proteome analysis proved to be a powerful tool for the identification of novel staphylococcal antigens, which provide a basis for rational vaccine design.     

Cell mediated immunity in Chagas' disease. Trypanosoma cruzi antigens induce suppression of the in vitro proliferative response of mononuclear cells.

The partial suppression of the cell-mediated immune response by Trypanosoma cruzi antigens in patients with Chagas' disease is demonstrated in a costimulation assay with T. cruzi antigens and Mycobacterium tuberculosis purified protein derivative (PPD) or Tetanus toxoid (TT). Mononuclear cells from 13 patients with chagasic infection without evidence of heart disease, 10 patients with chagasic cardiomyopathy and 7 healthy blood bank donors were stimulated with antigen A (autoclaved epimastigotes), PPD, TT, PPD + A, PPD + TT and TT + A. The average percentage of suppression induced by costimulation of mononuclear cells with PPD and antigen A was 47.1% in patients with chagasic infection without heart disease (INF), 38.8% in patients with chagasic cardiomyopathy (CDM) and 23.3% in healthy controls. Similar values were observed when living trypomastigotes were used. A costimulatory study with PPD and TT, PPD and A and TT and A was carried out in 8 patients with chagasic infection, in order to evaluate the possibility that this difference could be due to a nonspecific inhibitory effect. The mean suppression induced by TT + PPD was -8.9, with TT + A was 52.7 and with PPD + A was 50.1. The data reported show that T. cruzi antigens induce a specific suppression of the proliferative response of mononuclear cells, that might be relevant to the persistence of the parasite in the host.