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Extraction, separation, and partial characterization of Brucella abortus antigens.

Brucella abortus isolates strain 19 (a vaccinal strain) and 458 (a virulent field isolate) were analyzed for antigenic differences. Whole cell preparations were extracted with detergents, salt solutions, and phenol-acetic acid-water (PAW). Antigens were separated by starch block electrophoresis and sucrose density gradient centrifugation, serotested, and chemically analyzed. Six distinct precipitin lines were observed for the PAW extract against hyperimmune bovine antisera. With sucrose density centrifugation or starch block electrophoresis, antigens were separated into (i) complement-fixing (CF) antigens and (ii) antigens that did not fix complement but formed precipitin bands. Reciprocal CF tests with purified CF antigen could not differentiate between the antigens from either isolate of Brucella.

Animals↗

Diagnosis of infectious bronchitis (IB) by examination of tracheal mucus for IB-precipitating antigens.

The use of tracheal exudates for detecting precipitating infectious bronchitis (IB) antigen by the agar-gel precipitin (AGP) technique is described. Of 47 experimentally infected tracheas, 24 gave positive precipitin lines between the second and 12th day postinfection; 9 out of 18 tracheas from birds from natural outbreaks of IB were also positive. Tests can be read within 18-24 hr. For best results, several antisera or an antiserum at various concentrations should be used.

Animals↗

Preliminary immunologic studies of ciguatera poisoning.

Preliminary immunologic studies of ciguatera poisoning were performed with serum samples from patients and extracts from both toxic and nontoxic fish. Counterimmunoelectrophoresis disclosed precipitin lines with toxic fish extracts and effectively discriminated between samples established as toxic or nontoxic on the basis of human toxic reactions and by an in vivo mouse bioassay. However, it is not currently possible to conclude that affected individuals had specific antibody to ciguatoxin, since putative immune and nonimmune serum samples gave equally clear precipitin reactions with toxic extracts.

Animals↗

Animal studies of potential chronic lung disease of workers in swine confinement buildings.

The pathologic changes in laboratory animals housed in a swine confinement building were studied to predict potential chronic health effects on persons working in these buildings. Rabbits and guinea pigs were maintained for 12 months either in a confined nursery-grower unit (test animals) or in conventional laboratory animal housing (controls). Necropsies and histopathologic examinations were performed on animals that died prematurely or were killed at termination of the study. Blood serum from the animals was examined for antibodies to extracts from swine confinement house dust. The confinement atmosphere was monitored for environmental pollutants. Concentrations of gases and dust in the air of the confinement building were comparable to those found in a typical swine confinement building. Pulmonic lesions seen in the test rabbits and guinea pigs included diffuse interstitial histiocytic pneumonia. Tracheal and nasal turbinate lesions included epithelial hyperplasia and metaplasia, with submucosal infiltration of plasma cells and heterophils. Blood from the test animals contained serum precipitins to dust extract from confinement houses. The presence of precipitins combined with the microscopic appearance of the lungs indicated that an immunologic process, such as hypersensitivity pneumonitis, may be used to explain the basis of the observed lung lesions. The tracheal and turbinate lesions are best explained as a reaction to a chronic low-grade irritation.

Agricultural Workers' Diseases↗

Allergic bronchopulmonary aspergillosis: a case report and comment on reliability of testing sources.

A case of allergic bronchopulmonary aspergillosis (ABPA) is presented. Difficulty in establishing the diagnosis occurred because of a false-negative precipitin result obtained from a commercial laboratory. This would seem to support the contention that the paucity of ABPA cases reported in the United States is secondary to the low potency and variable composition of commercial antigens available for skin tests and immunodiffusion, resulting in false-negative skin and precipitin tests. Furthermore, positive tests to species of Aspergillus other than fumigatus heretofore considered non-pathogenic must now be considered as possible etiologic explanations.

Adult↗

A monoclonal IgM lambda macroglobulin with specificity for lacto-N-tetraose in a patient with bronchogenic carcinoma.

The serum of a patient with bronchogenic carcinoma was found to contain a monoclonal IgM lambda (IgMwoo) that precipitated with a precursor blood group glycoprotein containing I and i determinants. IgMwoo did not agglutinate O cells in the cold or at room temperature, and by quantitative precipitin and precipitin inhibition assay its specificity was shown not to be to the I and i determinants. IgMwoo reacted best with lacto-N-tetraose, DGal beta 1 leads to 3DGlcNAc beta 1 leads to 3DGal beta 1 leads to 4DGlc, and was specific for the non-I or non-i determinant dGal beta 1 leads to 3DGlcNAc beta 1 leads to 3DGal-moiety present as a distinct chain on precursor glycoproteins containing I and i determinants. Human ovarian cyst blood group A and B glycoproteins were inactive, but removal of the outer tier of sugars involved in A, B, and H specificity exposed this non-I or non-i determinant as well as the determinant reacting with anti-I Ma. IgMwoo was neither a cold agglutinin nor a cryoglobulin. It precipitated with precursor blood group glycoproteins somewhat less at 37 degrees C than at 0 degrees C, the differences being ascribable to solubility.

ABO Blood-Group System↗

[Two surveys on farmer's lung disease and bird breeder's disease in the Pyrenees region].

Two different surveys, one strictly epidemiological in rural areas, the other retrospective and within hospitals, led to a better knowledge of the setting of the FLD and BBD in South Pyrenees region. In the country, more than 12% of patients had specific precipitins, 6% had either one or both diseases; women had anti-aviary precipitins more frequently than men. The retrospective hospital survey yielded 456 cases of patients admitted or consulting revealing numerous correlations between the different clinical, radiological and functional syndromes on the one hand and the serological positivity on the other. Serological diagnosis was most useful, as a wide variety of antigenic extracts was used (actinomycetes thermophiles, mouldy hay, bird droppings), mixed sensitizing was extremely frequent. The widely found positivity towards avian antigens seems specific to the South Pyrenees region.

Adult↗

[The suppressive effect of antilymphocyte globulin (ALG) on the formation of anti-ALG antibodies].

The quantitative follow-up of precipitin formation against IgG aids in investigating the question whether ALG when applied to the organism tends to suppress the immune reaction against ALG. Rabbits locally immunized with pig anti-rabbit ALG were repeatedly treated i.v. with the same ALG, and to control groups normal pig IgG and NaCl saline was administered. It was found that antilymphocytic antibodies greatly suppressed the precipitation formation against IgG molecules. In the later stages of application this effect became more pronounced, evidently due to the specific suppression induced by long-term administration of relatively high doses of antigen. A possible improvement in the prevention of precipitin formation in ALG treated patients, i.e. substitution of currently applied tolerogenic dose of normal IgG by a similar dose of ALG is suggested.

Animals↗

[Spot test: use of a micromethod with whole blood in allergy diagnosis].

A limiting factor for the use of some in vitro techniques in the allergological diagnostic (RAST, PRIST, Precipitins) in the pediatric field, was the amount of serum needed for their carrying out, very difficult to obtain, especially in neonatology. In the present report the Authors record the results obtained by using a micromethod on the whole blood, called "Spot Test", which consists in the use of a particular base on which the blood drops are directly absorbed. The "Spot Test" showed the same sensitivity and reproducibility of the allergometric tests performed on the serum, therefore the correlation coefficients "r" for the RAST (r = 0.96), PRIST (r = 0.92) Precipitins (r = 0.89) showed to be highly significant for a close correlation between the two tests. Besides the "Spot test" turned out to be endowed, once the bases disc-blood dried with a stability at room temperature for several months (four months) without losing its sensitivity or specificity. This allows the possible sending of these bases to Specialized Centers without any particular problems of storage.

Humans↗

Allergic bronchopulmonary aspergillosis: serologic update for 1995.

Allergic bronchopulmonary aspergillosis (ABPA) is a complex disease occurring in 1-2% of patients with asthma involving hypersensitivity to Aspergillus species. The diagnosis is made correlating clinical history and supporting serologic tests. Over many years various serologic tests have been used to both diagnose and follow disease activity. Initial serology from patients with all four positive tests (total serum IgE, IgE and IgG antibody indices, serum precipitins to Aspergillus fumigatus) were compared with the patients' most recent serology. The total serum IgE, IgE antibody index and serum precipitins had the most initial positive tests which stayed positive throughout treatment. The IgG antibody index was the most inconsistent. There were no significant differences between all four tests. We concluded that all four serologic tests are important in the diagnosis of ABPA.

Antibodies, Fungal↗

[Induction of an allergic response of the delayed type against Salmonella abortus-ovis].

Studied were several of the conditions of inducing an allergy reaction of a delayed type in guinea pigs experimentally infected with Salmonella abortusovis as well as the correlation between the production of agglutinins and precipitins and the allergic reaction of a delayed type. It was found that a live culture of Salmonella abortusovis produced strongest allergic effects when injected into the pads. The injection (in the pads) of a killed culture, using no adjuvant, and the oral administration of a live culture did not sensibilize the animals. No correlation was established between the agglutinin and precipitin production and the allergic reaction of a delayed type.

Agglutinins↗

Metal working fluid-associated hypersensitivity pneumonitis: an outbreak investigation and case-control study.

Occupational exposure to bacterial or fungal antigens has been associated with hypersensivity pneumonitis (HP), an immunologically-mediated pulmonary disease. Between August 1995 and April 1996, 34 employees working in machining and assembly areas of an engine manufacturing plant were clinically diagnosed with HP. Of these, 20 employees met an epidemiologic case definition. In a case-control study, no exposure variables, including duration and intensity of metal working fluid (MWF) exposure, were statistically associated with an increased risk of disease. Neither cases nor controls demonstrated precipitin reactivity against unused samples of the seven MWF and two biocides used in the plant. HP cases had a significantly higher prevalence of positive precipitin reactions to used oil soluble and synthetic MWFs. Reactivity to used but not unused MWF suggests a biocontaminant, probably bacteria or fungi, is the causative antigen in the development of HP in this setting.

Alveolitis, Extrinsic Allergic↗

Induction of a specific (LM) protein in the submandibular gland of the rat by repeated amputation of the lower incisor teeth.

Chronic administration of the beta-adrenergic agonist isoproterenol (IPR) leads to marked hyperplastic/hypertrophic enlargements of the parotid and submandibular glands in rats and mice with concomitant changes in the composition of both the glands and the saliva. Conspicuous among the alterations of the submandibular saliva is the appearance of a 13,000 Mr protein, termed LM (large mobile) protein. Repeated amputation of the lower incisor teeth also causes enlargements of the major salivary glands in rats. In this study, we have compared the enlargements of submandibular glands of rats produced by IPR administration or teeth amputation with respect to the relative levels of the LM protein in gland extracts and saliva. Administration of IPR-HCl (40 mg/kg) twice daily for 5 days or amputation of the lower incisor teeth 3 times a week for 3 weeks resulted in a 2.2-fold increase in the weight of the submandibular gland. Amputation for one week led to a 1.4-fold increase in gland weight. Double immunodiffusion in agar antibodies against the purified LM protein gave a single precipitin line with gland extracts and saliva of IPR-treated and teeth-amputated rats, indicating immunological identity of the reacting antigens. No precipitin lines were seen with gland extracts or saliva of untreated rats. Immunoblots of pooled saliva obtained from IPR-treated or teeth-amputated rats revealed a single protein band of the same electrophoretic mobility in SDS-polyacrylamide gels when stained using anti-LM antibodies. The relative concentrations of LM protein in gland extracts and saliva were measured by a solid-phase enzyme-linked immunoabsorption assay using antibodies against the purified LM protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Antigenicity of a recombinant Ro (SS-A) fusion protein.

The antigenicity of the 60-kd human Ro (SS-A) synthesized in vitro from its complementary DNA as a beta-galactosidase fusion protein (beta-gal-Ro) was evaluated by Western blotting. In this analysis, almost all the anti-Ro (SS-A)-positive sera that bound beta-gal-Ro also bound affinity-purified 60-kd human Ro (SS-A) (P less than 0.005). Three of the 27 anti-Ro (SS-A) precipitin-positive sera, however, did not show reactivity on Western blot analysis, which suggests that in some sera, antigenicity to Ro (SS-A) is destroyed by denaturation. Of the 22 sera that were reactive with beta-gal-Ro, 2 were not reactive with affinity-purified human Ro (SS-A). Two serum samples that did not react with beta-gal-Ro were also reactive with affinity-purified human Ro (SS-A). Nevertheless, except for a small percentage of Ro (SS-A) precipitin-positive sera, the frequency of antibody binding to the fusion protein was similar to the frequency of binding to the purified antigen in Western blots. Recombinant Ro (SS-A) antigen may therefore be valuable in the serologic evaluation of anti-Ro (SS-A) autoantibodies.

Autoantigens↗

Dissociation of immune responses to the SS-A (Ro) 52-kd and 60-kd polypeptides in systemic lupus erythematosus and Sjögren's syndrome.

The cellular RNA particle SS-A (Ro) is a target of autoimmune response in many patients with Sjögren's syndrome (SS) and systemic lupus erythematosus (SLE). Recent immunologic, biochemical, and DNA cloning studies have shown that the SS-A particle consists of at least 2 polypeptide components, of 52 kd and 60 kd. Immunodiffusion analysis of 60 sera from patients with primary SS revealed 47 (78%) to be SS-A precipitin positive. Western blotting studies of the sera showed 3 groups of reactivities: 22 (47%) possessed autoantibodies against both the 60-kd and the 52-kd polypeptides, 19 (40%) reacted only with the 52-kd protein, and 6 (13%) were nonreactive in Western blots although positive in immunodiffusion. Fifty-one of 90 SLE sera (57%) were SS-A precipitin positive by immunodiffusion. In Western blots, 24 (47%) possessed antibodies against both the 60-kd and the 52-kd antigens, while 9 (18%) reacted only with the 60-kd protein. Eighteen (35%) were nonreactive by Western blot, although positive by immunodiffusion. Antibody to the 52-kd antigen without concomitant antibody to the 60-kd antigen was seen only in patients with primary SS, whereas antibody to the 60-kd antigen without concomitant antibody to the 52-kd antigen was seen only in SLE patients. Although antibodies to SS-A are detected in both SS and SLE, our findings show that there is dissociation of immune responses to the 2 component antigens of the particle, which may be evidence of different events initiating the autoimmune process in these diseases.

Antibody Formation↗

Acute familial myositis with a common autoimmune response.

In 1986, we diagnosed and treated 4 patients, all members of 1 rural family, who presented simultaneously with a polymyositis-like syndrome, anticytoplasmic antibodies (on HEp-2 cells), and precipitating antibodies of anti-Jo-1 and anti-PM-Scl specificities. Serum samples from these patients reacted by immunodiffusion against serum from a rodent that had been caught in the patients' house. The precipitin line showed complete immunologic identity with anti-Jo-1. To study the specificity of this serum-serum reaction, 2 mice (Mus musculus) and 1 rat (Rattus rattus) from the neighboring areas of the patients' house were caught and killed. Sections of the liver, kidney, spleen, and skeletal muscle were processed for histopathologic examination and for direct and indirect immunofluorescence studies of the patients' sera. The rodents' sera were tested by immunodiffusion against serum samples from 25 patients with systemic lupus erythematosus, 11 with rheumatoid arthritis, 6 with mixed connective tissue disease, and 11 with various other rheumatic diseases, as well as 16 healthy controls. A serum-serum precipitin reaction was noted between the rodents' sera and sera from 2 of the patients with polymyositis-like syndrome. A distinct, but weaker, reaction was found with 2 lupus patients' sera (8%), and with 1 healthy control serum (6%). The rodents' sera were antinuclear antibody positive by indirect immunofluorescence on HEp-2 cells. These results, suggest that some rodents could be carriers of an as-yet-unknown transmissible agent that, in susceptible individuals, induces clinical and serologic manifestations similar to those of polymyositis.

Acute Disease↗

Antibodies to vesicular stomatitis virus proteins in patients with systemic lupus erythematosus and in normal subjects.

OBJECTIVE: To determine the presence of antibodies that bind vesicular stomatitis virus (VSV) proteins in systemic lupus erythematosus (SLE) patient sera, as well as to examine any relationship between antibodies binding the viral nucleocapsid (N) protein and anti-Ro autoantibodies. METHODS: Eighty SLE patient sera, 80 normal control sera, 47 rheumatic disease patient control sera, and 9 sera from Panamanian subjects (6 of whom were infected with VSV) were evaluated by immunoblot of purified VSV proteins. RESULTS: Findings on immunoblots of the Panamanian subjects' sera were consistent with histories of endemic exposure to VSV, as determined by virus neutralization assays. Significantly different binding to viral proteins was seen in the group of 80 SLE patients compared with the normal control group and with the rheumatic disease control group, by a variety of measures. For example, most of the reactivity in the SLE patient sera included binding to the internal viral matrix (M) and N proteins, while the reactivity in the normal control sera was mostly against the surface viral glycoprotein (G) protein alone or the N protein alone. Within this SLE patient cohort, associations were found between binding to N protein and anti-Ro precipitins and between binding to M protein and anti-nuclear RNP autoantibody precipitins. CONCLUSION: The data are consistent with the original specific hypotheses of a relationship between 60-kd Ro autoantigenicity and VSV, as well as with the suggestion that a rhabdovirus may be important in SLE. They do not, however, make possible any conclusions concerning the role of rhabdoviruses in the development of SLE or of anti-Ro autoimmunity.

Antibodies, Viral↗

Immunochemical characterization of hepatitis B e antigen subtypes, HBeAg/1 and HBeAg/2, in sera of hepatitis B virus carriers.

The interrelation between HBeAg subtypes, HBeAg/1 and HBeAg/2, in sera was examined immunochemically. The detection of HBeAg subtypes in immunodiffusion (ID) depended upon the amount of HBeAg determined by reversed passive haemagglutination (RPHA), ie, the titres of HBeAg in sera positive for both HBeAg/1 and HBeAg/2, positive for only HBeAg/1 and negative for both HBeAg/1 and HBeAg/2 were 2(9.8) +/- 1.5, 2(7.0) +/- 1.6, and 2(5.6) +/- 1.3, respectively. When the sera belonging to the latter two groups were concentrated up to 2(10) RPHA titre, the precipitin line corresponding to that of HBeAg/2-anti-HBeAg/2 was visualized in ID. Monoclonal anti-HBeAg antibody that absorbed only the precipitin line of HBeAg/1-anti-HBeAg/1 in ID was prepared for the characterization of HBeAg subtypes. A linear correlation (r = 0.91) between titres of HBeAg determined by the RPHA cells prepared with monoclonal and polyclonal antibodies was found in almost all HBeAg-positive sera. The reactivities of this monoclonal anti-HBeAg antibody to both HBeAg/1 and HBeAg/2 were demonstrated in affinity chromatography experiments using a Sepharose 4B column conjugated with this antibody. These results suggest that both HBeAg/1 and HBeAg/2 are constantly present in HBeAg-positive sera and that they are closely associated. Based upon these results, a hypothetical model for the elucidation of the immunological relationship between HBeAg/1 and HBeAg/2 is proposed.

Animals↗