High levels of IgA-containing circulating immune complex and secretory IgA in Kawasaki disease

Sera of patients with Kawasaki disease were studied for the levels of IgA-containing (C3-fixing) circulating immune complexes (IgA-CIC), IgG-containing (IgG-)CIC, total IgA, secretory IgA, and complement component (C3) by means of enzyme-linked immunosorbent assays or single radial immunodiffusion methods. There was significantly high level of IgA-CIC, but not IgG-CIC. The levels of total IgA, secretory IgA, and C3 were significantly elevated. Significantly high levels of secretory IgA were found in 22 (51%) of 43 patients. The proportion of secretory IgA to total IgA also increased. These abnormalities in the IgA system may play a role in Kawasaki disease.     

Immunochemical and acarological study of mites

The data derived in the study of Dermatophagoides pteronyssinus by microscopic and immunological assays (double radial immunodiffusion in 2 modifications and indirect rat mast cell degranulation) are provided. The immunochemical assays permitting the detection of mites in 93.3% of cases have been shown to compare very favourably with the microscopic techniques that make it possible to detect mites only in 41.7% of cases.     

A comparative evaluation of the biological action of allergens and allergoids prepared from plant pollen by the double radial immunodiffusion method

Antisera to allergens and allergoids prepared from timothy, orchard grass, birch and wormwood pollen have been obtained and used in the double radial immunodiffusion test. The preparations of the allergoid row have been found capable of inducing immune response in laboratory animals (rabbits). Both forms of pollen preparations, allergens and allergoids, have been shown to possess common antigenic determinants reacting with antibodies present in antisera to allergens and allergoids. The absence of identity in the ratio of manifestations of gel precipitation reactions for allergoid with respect to the initial forms of allergens of individual pollen preparation has been noted.     

Salivary IgA is not a reliable indicator of upper respiratory infection in collegiate female soccer athletes

It has been shown that mucosal immunity measures such as salivary immunoglobulin A (s-IgA) can be affected by sport activities and has resulted in an increased susceptibility to infection. However, there is limited research that has evaluated the change in s-IgA throughout a full sport training season. The purpose of the study was to evaluate the change in s-IgA levels and incidence of upper respiratory infection in the National Collegiate Athletic Association Division I level female soccer athletes compared to age matched controls over an entire sport training season. Saliva samples were collected from 12 randomly selected female collegiate soccer athletes and 8 age-matched controls. Samples were collected bimonthly from the athletes' pre-and post-sport training sessions and pre- and post-90-minute sedentary period for the controls. Analysis showed there was a significant (p < 0.05) group × time interaction in total protein (TP) for collections 1 and 4 and a significant (p < 0.05) group × time interaction in s-IgA/TP for collections 2 and 3. There was no significant difference (p > 0.05) between athletes and controls for s-IgA or total symptom days (TSDs). Furthermore, there was no significant correlation between absolute s-IgA and TSDs or s-IgA/TP and TSDs throughout the sport training season. The large range of measurable levels for s-IgA at the different time points for athletes and controls and the lack of relationship between s-IgA levels and TSDs indicate that s-IgA is not an appropriate measure to determine an athlete's susceptibility to during a training season.     

Physiological responses induced by pleasant stimuli

The specific physiological responses induced by pleasant stimuli were investigated in this study. Various physiological responses of the brain (encephaloelectrogram; EEG), autonomic nervous system (ANS), immune system and endocrine system were monitored when pleasant stimuli such as odors, emotional pictures and rakugo, a typical Japanese comical story-telling, were presented to subjects. The results revealed that (i) EEG activities of the left frontal brain region were enhanced by a pleasant odor; (ii) emotional pictures related to primitive element such as nudes and erotic couples elevated vasomotor sympathetic nervous activity; and (iii) an increase in secretory immunoglobulin A (s-IgA) and a decrease in salivary cortisol (s-cortisol) were induced by rakugo-derived linguistic pleasant emotion. Pleasant emotion is complicated state. However, by considering the evolutionary history of human being, it is possible to assess and evaluate pleasant emotion from certain physiological responses by appropriately summating various physiological parameters.     

The effect of strain differences on the assay of rabies virus glycoprotein by single radial immunodiffusion

Antigenic differences between rabies virus strains used for vaccine manufacture can be demonstrated using monoclonal antibodies. We have shown that these differences are sufficiently large to affect the potency values of vaccines measured in single radial immunodiffusion (SRD) assays if the reference and test vaccines are antigenically heterologous. The production of reagents for use in SRD assays for each strain of rabies virus should be considered.     

Salivary immunoglobulin A response to a collegiate rugby game

Transient fluctuations in immune function after heavy exercise have been linked to an increased incidence of infection in athletes. Several parameters of immunity, including salivary immunoglobulin A (IgA), are affected by heavy exercise in the laboratory setting. However, few observations have been made during true competition. We tested the hypothesis that salivary IgA levels will be decreased after a collegiate rugby game. Saliva samples obtained from 16 men's college rugby players before and after an 80-minute regulation rugby game were analyzed for total volume, IgA, total protein content, and osmolality. Salivary IgA was expressed relative to secretion rate (s-IgA), osmolality (IgA-Osm), and total protein (IgA-Pro). No significant pregame-postgame changes in salivary IgA were observed (s-IgA: -13%, IgA-Osm: -16%, IgA-Pro: +10%). These data indicate that strenuous physical activity, such as a competitive rugby game, does not affect IgA levels. More study on the immune response to athletic competition is needed.     

Construction and use of a template block for radial immunodiffusion.

Details for design and construction of template blocks for the increased sensitivity of radial immunodiffusion (RID) assays are described. The sensitivity of the template RID was 10-fold higher for quantifying albumin and 5-fold higher for IgG than the conventional RID with wells cut into the agarose. The system described in this communication resulted from the unit which provided the most consistent results from a variety of trials with different block thicknesses, chamber sizes and depths, and chamber arrangements.     

A collaborative study on the use of single radial immunodiffusion for the assay of rabies virus glycoprotein

The single radial immunodiffusion (SRD) technique has been applied to the assay of the glycoprotein content of rabies vaccines produced in cell cultures. Fourteen laboratories in seven countries participated in a collaborative study to evaluate the reproducibility of the SRD technique; some laboratories also examined vaccines in the mouse protection (NIH) test and by enzyme immunoassay. Good agreement was found between potency estimates using the SRD technique: the geometric coefficients of variation for combined potency estimates of all laboratories were about 10%. SRD assays appear to have a role for the in vitro assay of antigen content of vaccine and could complement results obtained in in vivo assays which are subject to wide variability.     

Liposomes as a Mucosal Adjuvant System: An Intranasal Liposomal Influenza Subunit Vaccine and the Role of IgA in Nasal Anti-Influenza Immunity

Abstract

Liposomes exhibit potent immunoadjuvant activity in a variety of experimental vaccine formulations. We have investigated the mucosal adjuvant activity of liposomes in an influenza subunit vaccine. Mice were immunized intranasally (I.N.) with the major surface antigen of influenza virus, hemagglutinin (HA), mixed with negatively charged liposomes. Inclusion of the liposomes in the vaccine resulted in a marked stimulation of the serum IgG response against the antigen. In addition, the liposomal preparation, but not the antigen alone, induced a significant secretory IgA (s-IgA) response, not only in the lungs and nasal cavity, but also at the mucosa of the urogenital tract. The adjuvant activity of the liposomes appeared to be independent of a physical association of the antigen with the liposomes: Stimulation of antibody responses was observed even when liposomes and antigen were administered separately in time. Serum IgG and local s-IgA responses to I.N. immunization with the liposomal vaccine were comparable to the corresponding responses induced by an influenza infection. Mice immunized with the liposomal vaccine or mice recovered from an influenza infection were completely protected from (re)infection. Protection from nasal infection was abrogated by treatment of the mice with an anti-IgA antiserum, while anti-IgG had no effect, indicating that s-IgA plays an essential role in nasal anti-influenza immunity.     

Comparability of some serum protein determinations by radial immunodiffusion, laser nephelometric and turbidimetric assays employing Q-antisera SEVAC

Quantitation of IgG, IgA and IgM immunoglobulins and C3, C4 components in human serum samples by the radial immunodiffusion technique and by the nephelometric and turbidimetric assays was compared using the linear regression analysis method. Comparisons of the two methods run in polyethylene glycol showed close agreement between methods and a relatively high degree of correlation between the parameters studied. Compared to the radial immunodiffusion technique, nephelometry and turbidimetry gave good correlation between parameters, but the agreement between tests was worse, especially in the case of C3 component determinations in fresh samples of patients' sera. All tests were carried out using the Q-antisera and Control human serum preparations SEVAC.     

The composition of immune sera against Neisseria meningitidis studied by high-performance liquid chromatography

The comparative study of the composition of immune rabbit sera to N. meningitidis, as well as nonimmune sera, has been made by the methods of HPLC and radial immunodiffusion. The quantitative evaluation of the main serum proteins by the two methods has shown the coincidence of the results yielded by these methods. To study the total level of IgM and IgG in the sera under study, a simple and rapid HPLC technique is proposed. The study of the stability of sera during storage (at 4-6 degrees C) has revealed that immune sera show greater stability during storage under such conditions in comparison with sera obtained from nonimmune animals.     

Annotations on the measurement of liver ferritin, especially in the rat.

A radial immunodiffusion method (RID) to measure liver ferritin protein has been reevaluated. The RID has been compared with an electroimmunoassay (EIA) and with an enzyme-linked immunoassay (ELISA). The RID and ELISA provided similar results; the RID measured more ferritin protein in liver homogenates as compared with the EIA. After incubation of liver homogenate with deoxycholate (30 mg/ml) a slight increase of the ferritin concentration is detectable.     

Evaluation of the Lon-deficient Salmonella strain as an oral vaccine candidate

We evaluated the efficacy of CS2022 (the Lon protease-deficient mutant strain of Salmonella enterica serovar Typhimurium) as a candidate live oral vaccine strain against subsequent oral challenge with a virulent strain administered to BALB/c and C57BL/6 mice. CS2022 persistently resided in the spleen, mesenteric lymph nodes, Peyer's patches, and cecum of both strains of mice after a single oral inoculation with 1 x 10(8) colony-forming units. Finally, CS2022 almost disappeared from each tissue sample by week 12 in BALB/c mice, whereas CS2022 still resided in each tissue type at week 12 after inoculation of C57BL/6 mice. A significant increase in the serovar Typhimurium lipopolysaccharide-specific secretory immunoglobulin A (s-IgA), as measured for one of the mucosal immune responses, was detected in bile and intestinal samples of both strains of immunized mice at week 4 after immunization. In addition, the expression of gamma interferon mRNA in the spleens of both strains of immunized mice, especially those of C57BL/6 mice, was significantly increased at week 4 after immunization and was boosted during the following 5 days after the challenge was administered to the mice. Furthermore, peritoneal macrophages isolated from immunized mice at week 4 after immunization exhibited an increase in intracellular killing activity against both virulent and avirulent Salmonella. The present results suggested that salmonellae-specific s-IgA on the mucosal surfaces induced by immunization with CS2022 generally prevented mice from succumbing to an oral challenge with a virulent strain. Simultaneously, CS2022 promoted the protective immunity associated with macrophages in both strains of mice.     

Calibration line determination of an heterologous antigen in radial immunodiffusion

Using known principles of radial immunodiffusion it is shown experimentally that an heterologous antigen may be quantified using an homologous antigen-antibody system. The proteins in which this is demonstrated are human serum albumin (the homologous antigen) and the plasma albumins of baboon (Papio hamadryas) and rhesus monkey (Macaca mulatta). Purification of the heterologous antigen is not required. The system also gives a measure of the degree of cross-reactivity between the homologous and heterologous antigens.     

Measurement of hemolytic complement and the third component of complement in nonhuman primates.

Complement, determined by hemolytic assay, and the third component of complement (C3), determined by radial immunodiffusion assay, were measured in nine nonhuman primate species. The species studied were the titi (Callicebus mollach). The sooty mangabey (Cercocebus atys), the thick-tailed galago or bushbaby (Galago crassicaudatus panganiensis), the crab-eating monkey (Macaca fascicularis), the rhesus monkey (Macaca mulatta), the bonnet monkey (Macaca radiata), the stumptailed macaque (Macaca speciosa), the yellow baboon (Papio cynocephalus), and the black-and-red tamarin (Saguinus nigricollis). Both sheep and bovine erythrocytes were used in the hemolytic complement assays. With the sheep erythrocyte system, sera from four species (yellow baboon, sooty mangabey, bonnet monkey, black-and-red tamarin) had similar titers with both antibody sensitized and non-sensitized erythrocytes. In contrast, the titers obtained using sensitized bovine erythrocytes was always higher than the values obtained using non-sensitized bovine erythrocytes. In all species, the titers for non-sensitized sheep erythrocytes was higher than the titer for non-sensitized bovine erythrocytes. When the species were compared for cross reactivity using the radial immunodiffusion assay for human C3, the rhesus monkey showed the strongest cross reaction; the thick-tailed galago, a prosimian, showed no detectable cross reactivity; and the other species examined showed intermediate degrees of reactivity.     

Restoration of IgA synthesis following transplantation of histocompatible bursa cells into cyclophosphamide treated chicks.

High dose cyclophosphamide (CY) administration to newly hatched Line 6 subline 1 (L6₁) chicks resulted in agammaglobulinemic birds. Monitoring of natural agglutinins to rabbit erythrocytes and immunoglobulin class quantitation by radial immunodiffusion analysis indicated that transplantation with 7-day histocompatible bursal cells partially restored the bursa-dependent immune system, including synthesis of IgA. Additionally, the bursa of Fabricius in this strain of birds appears to be highly susceptible to CY treatment as evidenced by the development of a relatively long-term agammaglobulinemia in > 85% of the CY-treated chicks.     

Determination of NADPH-specific dihydropteridine reductase in extract from human, monkey, and bovine livers by single radial immunodiffusion: selective assay differentiating NADPH- and NADH-specific enzymes

It has been difficult to determine exactly NADPH-specific dihydropteridine reductase [EC 1.6.99.10] in samples which also contain NADH-specific dihydropteridine reductase [EC 1.6.99.7], because the latter enzyme interferes with the activity measurement of the former. We have devised a method to measure selectively the NADPH-specific reductase in crude extracts of bovine, human and monkey livers by the single radial immunodiffusion method using specific antiserum against the enzyme. This method makes it possible to determine the enzyme amount in 5 microliters of the 3-volume extracts of the livers. The amounts of NADPH-specific dihydropteridine reductase were calculated to be 0.252, 0.296, and 0.583 munits/5 microliter of the extracts of bovine, human, and monkey livers, respectively.     

IgA and IgA diphtheria antitoxin responses from human tonsil lymphocytes.

Human tonsil lymphocytes were stimulated with diphtheria toxoid and then cultured in a Marbrook culture system so that antibodies could be measured in the culture supernatant. Specific antibodies were measured with excess radiolabeled antigen and antisera specific for each immunoglobulin class. Good IgG and IgA diphtheria antitoxin responses have been obtained and responding culture supernatants were shown to neutralize toxin. The relationship between antitoxin response in vitro and immunization of donors with toxoid was investigated. It was found that at least two immunizations after the age of 6 months were necessary to prime the tonsils for an in vitro antibody response. The IgG and IgA in culture supernatants were demonstrated by immunodiffusion and were measured by radioimmunoassay. By sucrose density gradient ultracentrifugation, it was shown that 40% of the IgA produced in the cultures was greater than 7S. Evidence was obtained that neither the IgA nor the specific IgA antitoxin bears secretory piece. It appears that human lymphocytes from tonsils produce polymer IgA in vitro without secretory piece.     

Radial Immunodiffusion: a Simple and Rapid Method for Detection of Marek''s Disease Antigen(s)

A qualitative radial immunodiffusion technique is described which detects antigen(s) in feathers from live or dead chickens infected with Marek's disease herpesvirus. Antiserum, which is incorporated into a support medium, reacts with antigen(s) in the feather tip producing a radial precipitin ring. Antigen(s) was detected in 93.3% of experimentally inoculated chickens 21 days postinoculation and in 100% of infected birds subsequently tested through 6 weeks. No antigen was detectable in the feathers of uninoculated control chickens. The technique is simple and rapid to perform. Positive tests could be detected after 1 to 2 hours of incubation. Antigen detection by the radial immunodiffusion test correlated well with other criteria of infection. This technique should have application as a laboratory research tool and as an adjunct for a rapid flock diagnosis of Marek's disease.