Assessment of the specific activity of commercial allergen preparations made of house dust in the bacteriosorption reaction: a comparison with the results of the radioallergosorbent inhibition test

The activity of the commercial batches of house-dust (HD) allergens was compared in the inhibition of the radioallergosorbent test (RAST) and in the direct bacteriosorbent test (BST), detecting IgG to the antigens by adsorption on the complexes of whole staphylococcal cells containing protein A. BST was made with rabbit antiserum to HD allergen. This antiserum inhibited RAST by 76% and, therefore, contained antibodies to most of the allergenic determinants of HD. At the same time, no significant correlation between the activity of 15 batches of HD allergen was revealed in RAST inhibition and in BST with the above antiserum. Nevertheless, the exhaustion of the antiserum with a batch of HD allergen showing low activity in RAST inhibition, but high activity in BST made it possible to obtain BST results significantly correlating with the data resulting from RAST inhibition in two series of experiments.     

Characteristics of regression interrelations between the immune reactions to different staphylococcal strains in chronic inflammatory diseases of the upper respiratory tracts

Regressive interrelations between antibody titers to 13 staphylococcal museum strains in the blood serum, polypous fluid and saliva of patients with chronic polypous rhinosinusitis and chronic tonsillitis were studied. The presence of sharply defined positive interrelations between antibody titers in the blood serum and polypous fluid of patients with chronic polypous rhinosinusitis with respect to all staphylococcal strains under study and the absence of significant interrelations between antibody titers in the blood serum and saliva of patients with chronic tonsillitis were shown. The problem of the importance of positive or negative interrelations between individual staphylococcal strains is discussed.     

The creation of specific immunity to staphylococcal infection in newborn infants by the intranasal administration of adsorbed staphylococcal anatoxin

The possibility of enhancing specific immunity in newborn infants by the intranasal administration of adsorbed staphylococcal toxoid to infants with a high risk of staphylococcal infection in doses of 1 drop (0.05 ml) into each nostril during the first 7-9 years of their life. On days 7-9 the level of anti-alpha-toxin in the blood rose to 3.8 +/- 0.14 I. U./ml and remained sufficiently high 3-6 months later. When this method was used for the simultaneous immunization of mothers, their antitoxic titers were not as high as in newborn infants. No side effects were observed. In the control group, the titers of anti-alpha-toxin were low during the whole period of observation. Infants immunized by the proposed method had no staphylococcal infections both during the newborn period and within the first year of their life. In the control group, 8 cases of minor forms of purulent septic infection were registered during the newborn period, and in 2 infants umbilical staphylococcal sepsis was diagnosed.     

Alpha and beta chains of hemoglobin inhibit production of Staphylococcus aureus exotoxins

Prior studies suggest Staphylococcus aureus exotoxins are not produced when the organism is cultured in human blood. Human blood was fractionated into plasma and water-lysed red blood cells, and it was demonstrated that mixtures of alpha and beta globins of hemoglobin (as low as 1 mug/mL) inhibited S. aureus exotoxin production while increasing production of protein A and not affecting bacterial growth. Pepsin but not trypsin digestion destroyed the ability of alpha and beta globin to inhibit exotoxin production. Exotoxin production by both methicillin-resistant and methicillin-susceptible organisms was inhibited. Production of streptococcal pyrogenic exotoxin A by Streptococcus pyogenes was unaffected by alpha and beta globin chains but was inhibited when produced in S. aureus. Use of isogenic S. aureus strains suggested the targets of alpha and beta globin chains, leading to inhibition of staphylococcal exotoxins, included the two-component system SrrA-SrrB. delta hemolysin production was also inhibited, suggesting the two-component (and quorum sensing) system AgrA-AgrC was targeted. The alpha and beta globin chains represent promising molecules to interfere with the pathogenesis of serious staphylococcal diseases.     

Effect of terrilytin on the activity of nonspecific resistance factors during immunization with staphylococcal anatoxin and staphylococcal infection

Terrilytin and immobilized terrilytin enhance the activity and intensity of phagocytosis and increase the concentration of lysozyme in nonimmunized animals. Both preparations increase the production of antibodies to staphylococcal alpha-hemolysin, the titers of beta-lysins, the activity and intensity of the phagocytosis of bacterial cells by peripheral blood leukocytes in animals immunized with staphylococcal toxoid and challenged with live staphylococcal culture. In healthy animals terrilytin and immobilized terrilytin induce an increase in total proteolytic activity and in the activity of alpha-1-antitrypsin and alpha-2-macroglobulin, decreased as the result of staphylococcal infection.     

Formation of pollen-induced allergy in guinea pigs after specific immunotherapy with a bacterial allergen

The investigation was aimed at elucidating the process of the development of allergy to exoallergens (e.g., to linear-leaf wormwood pollen) in the body after desensitization by specific immunotherapy (SIT) with heterologous (staphylococcal) allergen. The study revealed that in staphylococcal allergy the subsequent development of pollen sensitization occurred with greater intensity, and SIT was found to exert no influence on this process. For the first time pollen allergy was found to produce an unfavorable effect on the quality and effectiveness of SIT with heterologous staphylococcal allergen, this effect consisting in the acceleration and intensification of the process leading to the recovery of sensitization to staphylococci. The importance of measures for limiting contacts with heterologous allergens and their elimination during SIT is emphasized.     

Effect of detergent and glycerin on the efficacy of nasal immunotherapy with allergens and on the level of the lymphocyte subpopulation in the organs of local immunity

Experimental mixed allergy to staphylococcal antigens in guinea pigs was treated by the intranasal administration of a staphylococcal allergen with a surfactant or glycerin added. The treatment was found to produce a hyposensitizing effect with respect to immediate and delayed hypersensitivity. The addition of glycerine enhanced this effect. At the same time the level of T-lymphocytes in the lungs and the lymph nodes of the respiratory tract returned to normal. Detergent used at a concentration of 2% abolished the hyposensitizing effect of the allergen, stimulated T-lymphocytes in the lymph nodes of the respiratory tract and the lungs; the number of T-suppressors decreased.     

Antibodies to Staphylococcus aureus teichoic acids in the pathogenesis of chronic osteomyelitis

The titers of antibodies to S. aureus cell-wall teichoic acids have been determined in 97 orthopedic and traumatic patients with purulent diseases, differing by the activity of the process, by means of the enzyme immunoassay. These antibodies appeared in the patients' blood in active osteomyelitic process of staphylococcal etiology.     

Inhibition of Leukocyte Migration by a Staphylococcal Factor

Cell wall mucopeptide isolated from virulent strains of Staphylococcus aureus has previously been found to potentiate subcutaneous staphylococcal lesions in mice. This cell wall fraction was found to inhibit the migration of polymorphonuclear leukocytes toward a chemotactic stimulus, as tested by the micropore filter chamber technique. A close correlation was shown to exist between in vivo "mouse virulence" of staphylococcal strains and the in vitro inhibition of leukocyte migration by the cell wall factor.     

The functional interchangeability of enterobacterial and staphylococcal iron chelators

The functional interchangeability of staphylococcal and enterobacterial iron chelators was investigated with an indicator system in which minimally effective concentrations of ethylene diamine di-ortho-hydroxyphenyl acetic acid (EDDA) were used to inhibit the growth of indicator strains in the depth of simple agar media by making the iron unavailable. Test colonies were then applied to the surface of the media to determine whether the indicator organisms, by utilising chelators from the test colony could obtain the required iron for growth, in its vicinity.Approximately 50% of staphylococcal strains, both S. aureus and S. epidermidis, reversed the inhibition of enterobacterial indicators, whereas almost all enterobacterial test strains, representing five genera, reversed the inhibition of the staphylococcal indicators. A purified preparation of the enterobacterial iron chelator enterochelin also reversed the inhibition of four out of the five staphylococcal indicator strains.     

Production of monoclonal antibodies to staphylococcal enterotoxin A.

Spleen cells from mice immunized with staphylococcal enterotoxin A were successfully fused with NS-1 mouse myeloma cells. Two of the four clones studied produced monoclonal antibodies to staphylococcal enterotoxin A in growth medium which showed titers of greater than 10(6) to 10(7) when tested by the indirect enzyme-linked immunosorbent assay. These monoclonal antibodies showed reactivity with enterotoxins A and E in the enzyme-linked immunosorbent assay. However, the reactivity was higher with enterotoxin A than with enterotoxin E. Nanogram quantities of crude staphylococcus enterotoxin A from Staphylococcus aureus growth were detected by the monoclonal antibodies in electroimmunoblots via autoradiography.     

Sensitization prevalence, antibody cross-reactivity and immunogenic peptide profile of Api g 2, the non-specific lipid transfer protein 1 of celery

Background

Celery (Apium graveolens) represents a relevant allergen source that can elicit severe reactions in the adult population. To investigate the sensitization prevalence and cross-reactivity of Api g 2 from celery stalks in a Mediterranean population and in a mouse model.

Methodology

786 non-randomized subjects from Italy were screened for IgE reactivity to rApi g 2, rArt v 3 (mugwort pollen LTP) and nPru p 3 (peach LTP) using an allergen microarray. Clinical data of 32 selected patients with reactivity to LTP under investigation were evaluated. Specific IgE titers and cross-inhibitions were performed in ELISA and allergen microarray. Balb/c mice were immunized with purified LTPs; IgG titers were determined in ELISA and mediator release was examined using RBL-2H3 cells. Simulated endolysosomal digestion was performed using microsomes obtained from human DCs.

Results

IgE testing showed a sensitization prevalence of 25.6% to Api g 2, 18.6% to Art v 3, and 28.6% to Pru p 3 and frequent co-sensitization and correlating IgE-reactivity was observed. 10/32 patients suffering from LTP-related allergy reported symptoms upon consumption of celery stalks which mainly presented as OAS. Considerable IgE cross-reactivity was observed between Api g 2, Art v 3, and Pru p 3 with varying inhibition degrees of individual patients'' sera. Simulating LTP mono-sensitization in a mouse model showed development of more congruent antibody specificities between Api g 2 and Art v 3. Notably, biologically relevant murine IgE cross-reactivity was restricted to the latter and diverse from Pru p 3 epitopes. Endolysosomal processing of LTP showed generation of similar clusters, which presumably represent T-cell peptides.

Conclusions

Api g 2 represents a relevant celery stalk allergen in the LTP-sensitized population. The molecule displays common B cell epitopes and endolysosomal peptides that encompass T cell epitopes with pollen and plant-food derived LTP.     

Bacteriosorption reaction on a smooth surface for detecting antibodies and antigens

The solid-phase technique for the detection of antibodies and antigens has been developed and named the bacteriosorption test. The test is based on binding staphylococci containing protein A with the Fc-regions of IgG-antibodies attached to antigens immobilized on polystyrene. The possibilities of this technique have been analyzed with the use of diphtheria toxoid, house-dust allergen and homologous rabbit antisera. In the detection of antibodies the proposed test is not inferior to the passive hemagglutination test, and its sensitivity in the detection of antigens by the sandwich technique reaches 0.05-0.1 micrograms/ml. The specificity of the technique has been experimentally confirmed by the inhibition of the reaction with soluble antigen and staphylococcal protein A. The variability factor of the technique does not exceed 10%.     

Proteomics and immunological analysis of a novel shrimp allergen,Pen m 2

Shellfish are a common cause of adverse food reactions in hypersensitive individuals and shrimp is one of the most frequently reported causes of allergic reactions. A novel allergen from Penaeus monodon, designated Pen m 2, was identified by two-dimensional immunoblotting using sera from subjects with shrimp allergy, followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of the peptide digest. This novel allergen was then cloned and the amino acid sequence deduced from the cDNA sequence. The cloned cDNA encoded a 356-aa protein with an acetylated N terminus at Ala2, identified by postsource decay analysis. Comparison of the Pen m 2 sequence with known protein sequences revealed extensive similarity with arginine kinase (EC 2.7.3.3) from crustaceans. Pen m 2 was purified by anion exchange chromatography and shown to have arginine kinase activity and to react with serum IgE from shrimp allergic patients and induce immediate type skin reactions in sensitized patients. Using Pen m 2-specific antisera and polyclonal sera from shrimp-sensitive subjects in a competitive ELISA inhibition assay, Pen m 2 was identified as a novel cross-reactive Crustacea allergen. This novel allergen could be useful in allergy diagnosis and in the treatment of Crustacea-derived allergic disorders.     

Measurement of IgG-blocking antibodies: development and application of a radioimmunoassay.

A radioimmunoassay for measuring blocking antibodies has been developed. We used the ragweed antigen E system to show that the same blocking antibodies (IgG) measured by inhibition of antigen-induced leukocyte histamine release were precipitated in the binding assay (r8 = 0.96, p less than 0.001), thus validating a widely applicable technique for measuring blocking antibodies. Binding of phospholipase-A (Phos-A), the major allergen in honey bee venom, was also shown to correlate significantly with inhibition of histamine release. Hymenoptera (insect) hypersensitivity was used as a model to demonstrate application of the binding assay. Sera obtained from patients undergoing whole body extract therapy contained negligible amounts of specific blocking antibodies. Significantly higher blocking antibody titers to both whole honey bee venom and Phos-A were measured in sera drawn from patients immunized with whole venom. The use of the binding radioimmunoassay should facilitate management of allergic disease processes in which blocking antibodies are thought to be protective.     

Analysis of the immunologic class of measles antibodies using protein A-producing staphylococci

The suspension of staphylocci containing protein A in their cell walls (strain Cowan 1) has been used for the determination of the immunological class of measles antibodies in the blood of measles patients and healthy persons. Adsorption with staphylococcal reagent is a simple and precise method for the detection of IgM which cannot be detected at equal or higher IgG titers when the samples under study are treated with reducing substances similar to mercaptoethanol. The advantages given by the use of protein A preparations for differentiating the nature of serum antibodies are shown, which is essential for the diagnosis and epidemiological analysis of infectious diseases.     

Enhancement of antitoxic immunity in newborn infants by peroral administration of donor antistaphylococcal immunoglobulin

The possibility of enhancing specific immunity by the oral administration of homologous antistaphylococcal immunoglobulin in a dose of 50 I. U./kg b. w. before the first feeding was shown in 75 newborn infants with a high risk of staphylococcal infection. 24 hours after the first administration of Ig the titer of staphylococcal anti-alpha toxin in the blood rose from 0.68 +/- 0.05 I. U./ml to 2.9 +/- 0.14 I. U/ml, on day 7 this titer persisted at the level of 2.86 +/- 0.12 I. U./ml, and 3 months later the titer was 1.5 +/- 0.05 I. U./ml. No side effects were observed. In the reference group (50 infants) antitoxic titers remained low. No suppurative-septic diseases were observed in the test group within 3 months, while in the controls, focal forms of staphylococcal infection (12 cases) and sepsis (1 case) were registered.     

The results and characteristics of the mupirocin (Bactroban) sanative treatment of intranasal Staphylococcus carriers in a large hospital]

The action of mupirocin as a nasal ointment (Bactroban) was studied on intranasal carriers of the hospital staphylococcal strains. The study included 37 medical workers from different and mainly problem units of the large general hospital. The tolerability of the ointment was good. After the Bactroban use no complications of the patients were recorded. The efficacy of Bacroban by the microbiological criteria in total amounted to 100 per cent. The eradication of methicillin resistant Staphylococcus aureus (MRSA) was observed in 93 per cent of the cases. A decrease of the level of the nasal passages dissemination by MRSA and methicillin resistant coagulase-negative staphylococci (MRSC) up to such low titers as 100 and 90 per cent was stated. No difference in the action of Bactroban on MRSA, MSSA and MRSC was noted. The bacteriological monitoring for 3 to 4 months revealed a change of the staphylococcal strains in 94 per cent of the cases, recolonization by the same staphylococcal strain in 19 per cent, recolonization by some another staphylococcal strains in 33 per cent and no recolonization in 14 per cent. A stable decrease of staphylococcal strains was possible with simultaneous Bactroban sanitation of all the bacterial carriers of the hospital or its isolated unit.     

IgM neutralizing antibody responses to human herpesvirus-6 in patients with exanthem subitum or organ transplantation.

The assay for detecting IgM neutralizing (NT) antibody activity to human herpesvirus-6 (HHV-6) was developed by using pretreatment of blood sample with staphylococcal protein A. The activity was mostly present in IgM fractions of serum but not in IgA fractions separated by ultracentrifugation. The assay was used for seroepidemiological studies for HHV-6 infection. In primary HHV-6 infection, IgM NT antibodies appeared 5 to 7 days after onset of exanthem subitum, reached maximum titers at 2 to 3 weeks, and tended to decline to undetectable levels after 2 months. In contrast, reactivation of HHV-6 observed in organ transplants showed somewhat greater degree of IgM NT antibody responses that persisted for 2 to 3 months and became undetectable 5 to 6 months after transplantation. The level and persistence of NT antibody titers measured by the conventional method was generally greater than those of the IgM titers. The prevalence of the IgM NT antibodies was examined in healthy individuals. The antibody was first detected at 4 to 7 months of age (5%), reached maximum level at 8 to 11 months (40%), and was detectable by 4 to 6 years (17%). A few (4 to 5%) of adolescents and adults were positive for the antibody.     

Influence of Food Microorganisms on Staphylococcal Growth and Enterotoxin Production in Meat

Forty-four microorganisms were studied for their influence on staphylococcal growth and enterotoxin production. Inhibition was found to be more common than stimulation. Two types of inhibition were observed: inhibition of staphylococcal growth, and inhibition of enterotoxin formation with no apparent effect on growth. By use of a plate test, 12 of the 44 food microorganisms were found to inhibit staphylococcal growth at 35 C. Of the 12, 3 also inhibited growth at 25 C. No significant differences in inhibition were observed with the 15 strains of enterotoxigenic staphylococci. In meat slurries, inhibition of staphylococcal growth was found to be greater at 25 C than at 35 C. Results on inhibition obtained from the plate test could not be correlated with the effect of the organisms in slurries. Environmental conditions were found to affect markedly the influence of food microorganisms on staphylococci. Of the 44 food microorganisms studied, only Bacillus cereus was observed to stimulate significantly staphylococcal growth and enterotoxin formation. Stimulation was more pronounced with Staphylococcus aureus 196E than with other strains of enterotoxigenic staphylococci. Bacillus megaterium and Brevibacterium linens were inhibited by staphylococci. These organisms were completely inhibited when inoculated in mixed cultures with staphylococci. In pure cultures, good staphylococcal growth was found to be accompanied by enterotoxin production; however, in the presence of food microorganisms, good staphylococcal growth occurred without the formation of detectable levels of enterotoxin A.