Experimental hypersensitivity pneumonitis: influence of Th2 bias

Cultured murine CD4+ cells from Saccharopolyspora rectivirgula sensitized C3H/HeJ (Th1 bias) donors can adoptively transfer murine experimental hypersensitivity pneumonitis (EHP). We sensitized BALB/c mice (Th2 bias) with S. rectivirgula, obtained spleen and lung associated lymph node (LALN) cells, cultured the cells with specific antigen, and attempted adoptive transfer of EHP. We also treated both C3H/HeJ and BALB/c donor mice with IL4 and anti-IFNgamma before exposure to S. rectivirgula and then cultured cells from both spleen and LALN before attempted transfer of EHP. We found that cultured spleen and lung associated lymph node cells can adoptively transfer EHP in both C3H/HeJ and BALB/c mice as demonstrated by infiltration of the recipient lungs with CD4+ lymphocytes. Treatment of both mouse strains with IL4 and anti-IFNgamma did not change the ability of cultured cells to adoptively transfer EHP. We conclude that EHP induced by S. rectivirgula can occur in animals with either a Th1 or a Th2 bias and is not altered by treatment with IL4 and anti-IFNgamma. This suggests that attributes of the antigen and not genetic background or cytokine environment at the site of initial sensitization determines the results of exposure to S. rectivirgula.     

Multiple protein extract microarray for profiling human food-specific immunoglobulins A, M, G and E

Existing food immunoglobulin (Ig) tests require large volumes of serum, are limited to one immunoglobulin class, are not amenable to high throughput analysis and only give a limited picture of the immunological response to food antigens. Conversely a new generation of Component Resolved Diagnostic systems using pure proteins is highly specific and totally dependent on the availability of the protein in its recombinant or natural origin form. Here we demonstrate a proof-of-concept of a microarray test based on protein extracts of food components. Our approach relies on innovations on three different fronts: the novelty of using arrayed food samples sequentially extracted with detergent and chaotropic agents, the ability to measure four different Ig classes simultaneously and the ability to analyse the generated data via a suitable bioinformatics/statistical analysis interface. This approach combines high numerical power of microarrays with automation, high throughput analysis and enables detailed investigation of the Ig profiles to food antigens. The prototype shown contains extracts of approximately 350 food ingredients that cover most of the food products found in the UK. Here we showed that the use of a sequential extraction technique to solubilise and then denature food samples has its benefits in the assessment of variations in antigenicity when tested with human sera. A patient dependent degree of class specificity was observed with human sera (IgG specificity correlates well with IgA>IgM>IgE). Besides generating a simultaneous profile for IgA, IgM, IgG and IgE the array system has shown good discrimination between challenge responders in atopic and non-atopic individuals. Poly- and mono-specific IgE responders were easily identified. The mathematical modelling of specific IgE content showed good correlations when compared with established IgE antibody testing assay (UniCAP). Although in its proof-of-principle stages, the immune profiling technique described here has the potential to provide unique insights into exposure/sensitization and establish relationships between specific immunoglobulin classes and subclasses against food protein antigens. In further developments, the immune profiling technique could also be extended to other related areas such as parasite and bacterial gut infection. Full analyses of large longitudinal and retrospective clinical trials are on going to determine the positive and negative predictive values of the technique.     

Diagnosis of influenza A virus infections by detection of specific immunoglobulins M, A, and G in serum.

The value of immunoglobulin M (IgM) detection in the early diagnosis of influenza A was examined in a prospective study during an outbreak of influenza A/Philippines/2/82 (H3N2) virus infection in February and March 1986. The IgM response was investigated in sera from 64 adults with influenzalike symptoms; we found a fourfold rise in titer or high titers (greater than or equal to 80) of influenza A virus antibodies by the complement fixation test. The IgM response, analyzed by the hemadsorption immunosorbent technique, was compared with the IgG and IgA responses analyzed by an indirect enzyme-linked immunosorbent assay and the hemadsorption immunosorbent technique, respectively. Antigen detection in nasopharyngeal secretions by immunofluorescence was performed for all patients on admission to hospital. Specific IgM was detected in 86% (55 of 64) of the patients with influenza A. In sera from 36% (18 of 64) of the patients it was detected already on admission. Influenza A virus antigen was detected in nasopharyngeal cells by immunofluorescence on admission in 53% (34 of 64) of the patients. A combination of immunofluorescence and IgM results gave a significantly higher diagnostic rate, 69% (P less than 0.01), on admission than did each of the two tests separately. An IgA serum antibody response was seen in 76% (48 of 64) of the cases but did not contribute to any increase in the diagnostic rate. IgM detection by the hemadsorption immunosorbent technique was found to be a valuable supplement for the diagnosis of influenza A in an early phase of the disease.     

Experimental hypersensitivity pneumonitis. Humoral and cell-mediated immune response of cattle to Micropolyspora faeni and clinical response to aerosol challenge.

Hypersensitivity pneumonitis was produced in calves, following repeated exposure to aerosols of Micropolyspora faeni both with and without prior systemic sensitization. The condition was associated with rising serum, nasal and broncho-alveolar antibody titers and with transient ability of peripheral lymphocytes to synthesize macrophage migration inhibition factor. Clinical response to aerosols of M. faeni suggest participation of reaction types, I, III and IV of Gell and Coombs.     

Measurement of immunoglobulins G, A, and M levels in B-lymphocytes culture

Nephelometric immunoassays were developed for human IgG, IgA, and IgM quantitation in B-lymphocytes culture media. They allowed measurement of immunoglobulin (Ig) levels over a broad range of concentrations with good accuracy and precision. The kinetics of Ig production in B-lymphocyte cultures was followed and the mean amount of each Ig was determined in six different samples after three days of culture. The nephelometric immunoassays reported here could be used to study, in vitro, the influence of various molecules (inhibitory or amplifying effect) on B-lymphocytes' functional capacities.     

Experimental hypersensitivity pneumonitis in the rat: histopathology and T-cell subset compartmentalization

The pathogenesis of hypersensitivity pneumonitis (HP) appears to depend largely on T-cell specificity and interactions with monocytes/macrophages. We studied T-cell subset populations, defined by surface membrane markers with putative functional correlates, present in lung parenchyma and bronchoalveolar spaces of sensitized LEW rats following acute and chronic inhalational challenges with antigen. Our initial hypothesis was that the CD4+ RT6- (TDH) subset, the effector cell of delayed hypersensitivity, would dominate in HP lesions and that CD8+ RT6+ (TS) or CD8+ CD45R+ (TS) subsets, constituting putative suppressor T-cell populations, would dominate in lungs of animals with resolving lesions. We found, however, a heterogeneous population involving all eight of the T-cell subsets that were evaluated. Percentages of the RT6+ phenotype diminished as T cells moved from peripheral blood to lung. Dominant numbers of T cells in acute HP included CD8+ RT6- (TCYT) and CD8+ CD45R- (TCYT) subsets, with putative cytotoxic T-cell activity, in addition to CD4+ RT6- (TDH) cells. We did not demonstrate increases in T suppressor cells as the disease waned.     

Serum and cerebrospinal fluid immunoglobulins M, A, and G in Japanese encephalitis.

A comparison was made of virus-specific immunoglobulin M (IgM), IgA, and IgG detected by capture or indirect enzyme immunoassay in serum and cerebrospinal fluid of patients with Japanese encephalitis. The IgM capture enzyme immunoassay was more sensitive than assays for other isotypes of viral antibody; IgM was detected in 75% of specimens collected less than or equal to 4 days after the onset of illness. Specific IgA was detected in both serum and cerebrospinal fluid; however, IgA levels were significantly lower than IgM levels.     

Solid-phase radioimmunoassay of immunoglobulins G, A and M: applicability in analysis of sucrose gradients

A simple and sensitive solid-phase radioimmunoassay for the detection of immunoglobulins G, A and M in sucrose gradients is described. The solid-phase consisted of immunoglobulins adsorbed to polystyrene tubes.Using buffers without detergent and ¹²⁵I-labelled sheep anti-rabbit IgG as radioligand, the assay was able to detect 0.8 ng per tube in the IgG assay and 1.6 ng per tube in the IgA and IgM assays. Standard curves with antigen dissolved in 10% and 32% sucrose were superimposable and did not deviate from standard curves with antigen dissolved in buffer without sucrose.Using these techniques on ultracentrifugation samples from patients with systemic lupus erythematosus, Schönlein-Henoch nephritis and IgA glorulonephritis it was possible to detect both immunoglobulin fragments and immunoglobulin aggregates at the same time without prior dialysis of the samples.     

Antiviral activity of colostrum and serum immunoglobulins A and G

Enteric virus-specific IgA and IgG present in paired human sera and colostrums were measured by the enzyme-linked immunosorbent assay (ELISA). Virus-specific IgA was present in all colostrums, but virus-specific IgG could not be detected. The reverse was true when sera were assayed. Most of these colostrums also neutralized either polio virus or reovirus, as did IgA, which was separated from a pool of colostrums by exclusion chromatography. No correlation could be made between levels of neutralizing and ELISA antibody titers in colostrums.     

Experimental hypersensitivity pneumonitis in the mouse: histologic and immunologic features and their modulation with cyclosporin A

A murine model of hypersensitivity pneumonitis (HP) was established with transnasally administered Thermoactinomyces vulgaris (Tv) bacilli. Bronchoalveolar lavage (BAL) of experimental animals revealed marked increase in the total cell, lymphocyte, and macrophage numbers; the findings were similar to those in human HP. The BAL lymphocytes were mostly Thy 1.2 positive. Lyt-1 positive cells predominated Lyt-2 positive cells. Anti-Tv IgG antibodies and delayed-type hypersensitivity footpad reactions against Tv were detected in animals with HP. Cyclosporin A (CyA), a potent immunosuppressive drug, had marked effects on the development of HP in this model. When CyA was administered throughout the course of Tv inoculations, the granulomatous pneumonitis was markedly suppressed, and an increase in BAL lymphocytes, Thy 1.2 positive cells, was suppressed. When CyA was administered only during the first half period of the Tv treatment, suppression of the disease was minimal; when CyA was administered in the latter half, both the HP lesions and the increase in BAL cell lymphocyte numbers were significantly suppressed. These results indicate that a series of transnasal administration of Tv in mice may provide a good model for human HP.     

Kinetics of specific immunoglobulins M, E, A, and G in congenital, primary, and secondary cytomegalovirus infection studied by antibody-capture enzyme-linked immunosorbent assay

Antibody-capture enzyme-linked immunosorbent assay (ELISA) using enzyme-labeled cytomegalovirus (CMV) nuclear antigen is a reliable and easily performed test suitable for routine use. As the serologic response to CMV infection may, however, vary considerably among patients, we have studied the kinetics of CMV-specific immunoglobulin M (IgM), IgE, IgA, and IgG antibodies in 352 sera from 61 patients by using antibody-capture ELISA and complement fixation (CF) tests. In a CMV mononucleosis group (n = 17), most patients had antibodies of all four immunoglobulin classes, but antibody levels decreased rapidly, with half the patients having a borderline-positive or a negative reaction for all classes, except IgG, 2 months after the appearance of symptoms. Twelve patients with a primary CMV infection after renal or bone marrow transplantation also developed all immunoglobulin-class antibodies. In only two patients did CMV IgM and IgE antibodies precede seroconversion of CF antibodies, and in one patient, these antibodies lagged months behind. Most patients had all classes of CMV antibodies, except IgA, for a year or more. Among 10 transplant patients with a secondary CMV infection, 50% had long-lasting IgM antibodies, and very few had IgE or IgA antibodies, but all had IgG antibodies to CMV. In 13 infected infants, the CMV-specific serologic response was also characterized by long-lasting IgM, IgE, and IgG antibodies. Two patients did not develop detectable IgM antibodies, and one of these did not show IgE antibodies either. The IgA response in infants as a whole was lacking; a few, however, were borderline positive. Of the nine acquired immunodeficiency syndrome patients with CMV infection studied during their last year of life, only one had antibodies in all four classes, the rest had only CF antibodies, and all except for one had IgG-class antibodies. All sera studied were also tested against a control antigen produced from noninfected cell nuclei. It was found that some patients developed antibodies to nuclear antigens in parallel with the rise in specific antibodies. The nonspecific antibodies occurred in all four classes, but most often they were of the IgM class. Addition of unlabeled control antigen to the conjugates was not always sufficient to abort this nonspecific reaction.     

Aqueous humor and serum immunoblotting for immunoglobulin types G, A, M, and E in cases of human ocular toxoplasmosis

The purpose of this study was to compare the local and systemic Toxoplasma-specific humoral immune responses in individuals with ocular toxoplasmosis (OT). To this end, paired aqueous humor and serum samples from 46 individuals with active OT and from 30 individuals without inflammatory eye disease (controls) were analyzed by immunoblotting for anti-Toxoplasma immunoglobulin G (IgG), IgA, IgM, and IgE directed against 20- to 120-kDa antigens. The presence in the aqueous humor of a unique band, or of at least three bands that were at least three times more intense in aqueous humor than in serum, was taken as evidence of local antibody production. IgG bands were detected in 98% of the aqueous humor samples, while IgA bands were detected in 76%, IgM bands were detected in 8%, and IgE bands were not detected in any. Evidence of local production of specific antibodies was found in 32 cases (70%) (IgG in 23 [50%]; IgA in 16 [35%]). In 10 instances (22%), routine laboratory tests were not indicative of OT. In 14 cases (30%), no local antibody production was detected by immunoblotting; 3 of these cases yielded evidence of local antibody production according to the Goldmann-Witmer coefficient. Local antibody production was revealed for 7 of the 30 controls (23%). Hence, the sensitivity of immunoblotting for IgG and IgA is 70%, and the specificity is 77%. We conclude that immunoblotting for local specific IgG and IgA supports the clinical diagnosis of OT in 70% of cases. In 22% of these, the diagnosis is not confirmed by other laboratory tests. Hence, immunoblotting increases the sensitivity of routine laboratory tests and should be considered for samples that register negative by such tests.     

Role of immunoglobulins G1 and G2 in anaphylactic shock in the guinea pig

Heating serum from actively sensitised guinea pigs did not remove its ability to sensitise recipient animals in vivo and parenchymal lung strips in vitro to anaphylaxis. Thermoresistant antibodies should thus account for the transferable sensitising effect, which persists for at least 9 days. IgG1 and IgG2, contained in the serum, were separated by affinity chromatography to determine the importance and the participation of these subclasses in passive anaphylactic shock. IgG1, present in smaller amounts than IgG2, was more effective in sensitising isolated lung strips. The intravenous administration of ovalbumin to guinea pigs, which had been injected with 0.8 mg/kg of IgG1 or 2 mg/kg of IgG2 9 days beforehand, induced an intense bronchoconstriction with leucopenia and moderate thrombopenia, suggesting an as yet undescribed role for IgG2 in passive tissue sensitisation. The use of mepyramine, an antagonist of the histamine H1 receptor, WEB 2086, an antagonist of platelet-activating factor, and nordihydroguaiaretic acid, a dual inhibitor of cyclooxygenase and lipooxygenase, alone or associated, demonstrated that the anaphylactic contraction of lung strips from guinea pigs sensitised by IgG1 is mediated by histamine and arachidonate derivatives, whereas that of lung strips from guinea pigs sensitised with IgG2 is mostly mediated by histamine. In addition, the association of the three potential antagonists slightly reduced the anaphylactic contraction of lung strips provided by guinea pigs sensitised by serum. Our results, using a sensitisation procedure considered until now to involve exclusively IgE antibodies, indicate that IgG1 and IgG2 are in fact the essential antibodies for passive anaphylactic shock in the guinea pig.     

Detection of specific anti-leptospiral immunoglobulins M and G in human serum by solid-phase enzyme-linked immunosorbent assay.

The enzyme-linked immunosorbent assay (ELISA) was used to detect leptospire-specific immunoglobulin M (IgM) and IgG in the sera of patients infected with leptospiral serovars hardjo, pomona, or copenhageni. All patients produced specific IgM and IgG detectable by ELISA. In contrast, only a few patients produced IgG agglutinins whereas all produced IgM agglutinins. The specificity and sensitivity of the test suggest that the ELISA anti-IgM technique is a suitable method for detecting leptospiral antibodies in human sera for diagnostic and epidemiological purposes.     

Selective reactivity of antibodies to human immunoglobulins G, M, and A with rubella virus proteins

Proteins of purified rubella virus were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and immunoblotted with human sera and immunoglobulin class heavy-chain-specific peroxidase conjugates. The levels of rubella antibodies in these sera were predetermined by the radial hemolysis test, the density gradient centrifugation method for immunoglobulin M (IgM) antibodies, and IgG-, IgM-, and IgA-specific enzyme immunoassays. In immunoblotting, rubella-specific IgG antibodies reacted with both envelope glycoproteins (E1 and E2) and the capsid protein (C). In contrast, rubella IgM antibodies reacted predominantly with E1, whereas the specific reactivity of IgA antibodies was directed mainly to the capsid protein. Purified IgM rheumatoid factor added to IgG-positive, IgM-negative serum did not give false-positive reactivity in the immunoblotting test as it did in solid-phase enzyme immunoassays. The immunoglobulin class-specific reactivities with the different viral proteins are expected to have diagnostic applications.     

Degradation of immunoglobulins A2, A2, and G by suspected principal periodontal pathogens.

Attention has recently been focused on immunoglobulin A1 (IgA1) protease production as a possible virulence factor of bacteria implicated in meningitis and gonorrhea. This report demonstrates that suspected principal etiological agents in destructive periodontal disease include bacteria capable of degrading IgA1, IgA2, and IgG. Representative strains of Bacteroides melaninogenicus subsp. melaninogenicus and Capnocytophaga cleaved IgA1 but not IgA2 in the hinge region to yield intact Fab and Fc fragments. All Capnocytophaga strains also cleaved IgG in the same way. The majority of strains of Bacteroides asaccharolyticus and B. melaninogenicus subsp. intermedius caused complete degradation of both IgA1 and polyclonal IgG. However, some strains left the Fc part of IgA1 intact. Several strains were also capable of completely decomposing IgA2 and S-IgA. Significant IgA-cleaving enzyme activity was detected in whole subgingival dental plaque collected from patients with destructive periodontal disease. The results indicate that colonization of the subgingival area by B. asaccharolyticus, B. melaninogenicus, and Capnocytophaga spp. can induce a local paralysis of the immune defence mechanisms, thereby facilitating the penetration and spread of potentially toxic substances, lytic enzymes, and antigens released by the entire subgingival microflora.