Expression, purification and immunochemical characterization of recombinant bovine beta-lactoglobulin, a major cow milk allergen

The immunological characteristics of a recombinant beta-lactoglobulin were studied using monoclonal antibodies, polyclonal antiserum and sera from allergic patients. Recombinant beta-lactoglobulin (rBLG) was expressed in Escherichia coli strain DH5alpha and purified as described previously [Cho et al. (1994) J. Biol. Chem. 269, 11 102-11 107]. The method has been modified by adding an immunoaffinity purification step. A quantity of 5-10mg of purified rBLG per liter of medium culture can be produced. rBLG shared the same molecular weight as the natural BLG (nBLG) and also possessed at least one intrachain disulfide bridge. In HPLC, rBLG appeared as a single peak, and the purity was estimated to be greater than 95%. All the monoclonal antibodies (mAbs) used in this study recognized different epitopes of the BLG and presented compatible binding. No differences could be detected between rBLG and nBLG when tested in a Western blot with rabbit polyclonal antiserum or with three mAbs that bound preferentially the reduced and S-carboxymethylated form of BLG. In a competitive enzyme immunoassay (EIA) using either a rabbit polyclonal antiserum or four mAbs that recognized conformational epitopes, we could not distinguish between rBLG or nBLG. In direct ELISA using nBLG or rBLG as the immobilized allergen, we measured a similar concentration of specific anti-BLG IgE in five sera from allergic patients. The results of this study indicate that we have obtained a rBLG with biochemical and immunological properties very similar to nBLG.     

Fine tuning of epitopic dominance induced by lung cancer on the IgG response to bovine betalactoglobulin: towards a paraneoplastic immune marker

BACKGROUND: Investigating the humoral immune response to mucosal antigens in patients with lung cancer, we have documented a preferential immunoglobulin G (IgG) binding to cryptic epitopes unmasked by the proteolysis of bovine beta-lactoglobulin (BLG). In contrast, IgG from healthy controls and patients with chronic bronchitis (COPD) bind preferentially to continuous epitopes presented on both native (n) and denaturated (d) forms of this antigen. The present study further characterized the differences in the epitope profiles recognized on BLG. METHODS: The capacity of individual sera from 65 lung cancer patients, tested before and after cancer removal for the patients with early stage lung carcinoma, 65 healthy controls, and 52 patients with COPD, to prevent the binding of pooled IgG fractions from each population as well as murine monoclonal antibodies (MoAb), specific for BLG, to solid phase bound antigen was evaluated in enzyme-linked immunoadsorbent assay using streptavidin-biotin technology. Some of these experiments were also performed with sera from 42 patients diagnosed with other cancers. RESULTS: Compared with control sera and sera from patients with other solid tumors, lung cancer patient sera showed distinct capacities to prevent the binding of murine MoAb as well as human pooled IgG fractions to n- and d-BLG. The inhibition capacities of lung cancer sera changed as soon as five weeks after cancer removal. CONCLUSIONS: The results indicate that the difference in epitope specificity exhibited by lung cancer sera is not restricted to cryptic epitopes, but also affects continuous and discontinuous epitopes, accessible only on the native antigen. A high level of binding discrimination between antibodies from the study populations is also observed at the level of the epitope. This deviation in the epitope specificity of antibodies changes soon after cancer removal, suggesting a tumor-dependent disturbance. Also documented in the Dermatophagoides pteronyssinus model, it opens the way to a new class of paraneoplastic immune markers for this malignancy, with, at first glance, a high specificity level.     

Detection of serum antibodies against Chlamydia pneumoniae by ELISA

Chlamydia pneumoniae causes pneumonia and other respiratory infections in children, adolescents and adults. We tried to evaluate the diagnostic value of detection of serum antibodies by ELISA for C. pneumoniae infections in Japanese children. Serum IgG, IgA and IgM antibodies to C. pneumoniae were determined by the microimmunofluorescence (MIF) test. Serum IgG and IgA antibodies were also determined by ELISA test kits. Results obtained by ELISA were compared with those obtained by MIF test. IgG antibody to C. pneumoniae was detected in 135 (39.5%) by ELISA and in 125 (36.5%) by MIF out of 342 sera from Japanese infants and children without respiratory infections (aged from 2 months old to 15 years old). IgA antibody to C. pneumoniae was detected in 129 (37.7%) by ELISA and in 117 (34.2%) by MIF out of 342 sera tested. Of 342 specimens 113 were IgG-positive by ELISA and MIF (sensitivity: 90.4%, specificity: 89.9%, r = 0.853). Of 342 sera 28 had IgG antibody titers of 1:256 and none had titers 1:512 or higher by MIF. Of 28 infants and children a total of nine were less than 4 years of age. On the other hand, of 342 specimens 99 were IgA-positive by ELISA and MIF (sensitivity: 84.6%, specificity: 86.7%, r = 0.769). Of 342 sera 16 had IgA antibody titers of 1:256 or higher by MIF. Of 16 infants and children, ten were less than 4 years of age. ELISA had excellent sensitivity and specificity relative to MIF test for detection of IgC and IgA antibodies to C. pneumoniae. It was suggested that C. pneumoniae infection in Japanese infants and children under 4 years of age was not infrequent.     

New laboratory guidelines for serologic diagnosis of Lyme disease: evaluation of the two-test protocol

Recent guidelines established by the Association of State and Territorial Public Health Laboratory Directors (ASTPHLD) and the U.S. Centers for Disease Control and Prevention (CDC) recommend the use of a two-test protocol for the serologic diagnosis of Lyme disease (LD). The two-test protocol relies on a sensitive screening test, which is followed by specific immunoglobulin M (IgM) and/or IgG immunoblotting (IB), depending on the date of disease onset, of all samples with equivocal and positive screening test results. We evaluated a commercially available IgM-IgG enzyme-linked immunosorbent assay (ELISA) and separate IB tests for IgM and IgG antibodies to Borrelia burgdorferi as candidate assays for the two-test protocol. Serum samples obtained from healthy controls (n = 29), from patients with diagnoses or laboratory findings associated with serologic cross-reactivity to LD (n = 24), and from patients with well-documented early- and late-stage LD provided by the CDC and the College of American Pathologists (n = 53) were examined to determine each assay's individual sensitivity and specificity. No false-positive results were detected among the healthy controls by either ELISA or IB, whereas four false-positive ELISA results were recorded within the cross-reactive group. None of these sera, however, were positive for either IgM or IgG reactivity according to IB band criteria. With regard to the patients with LD, we determined the sensitivity and specificity of the ELISA to be 96 and 100%, respectively, compared with the reference data provided for these specimens. When we compared our IB results with data from CDC, the assay sensitivity and specificity were 80 and 96.2%, respectively, for IgM and 81.8 and 95.8%, respectively, for IgG. Pursuant to this evaluation we assessed the suitability of the two-test protocol by performing a retrospective analysis using clinical history to define samples as positive or negative for LD. We determined clinical sensitivity and specificity for all study subjects (n = 112) to be 50 and 100%, respectively. A reduction in the clinical sensitivity of the two-test protocol was associated with a lack of antibody response or seroconversion in LD patients treated with antibiotics. We conclude that the CDC-ASTPHLD guidelines provide useful criteria for test performance and interpretation aimed at standardizing the serologic diagnosis of LD.     

Reproductive and productive performance in Chios ewes mated in spring or in autumn

To determine whether specific antibodies are present in PD, we used an enzyme-linked immunosorbant assay (ELISA) that identifies increased immunoglobulin (IgG) levels towards a synthetic substrate prepared by incubating ovalbumin with dopamine and copper sulfate. Altered absorption spectrum and specific chemical detection demonstrated quinone modification of the ovalbumin. This modified protein was demonstrated to react with serial dilutions of PD sera. A threshold dilution of 1:500 was subsequently used to screen sera from patients with PD (n = 21), amyotrophic lateral sclerosis (n = 7), Alzheimer's disease (n = 7) and other neurological disease controls (n = 7). The assay produced a positive result in 7/21 PD patients and 0/21 disease controls (P < 0.02, Kruskal-Wallis test). Further testing of sera from untreated PD patients (n = 6) identified one positive sample. Thus, a subset of Parkinson's disease (PD) patients has immunoglobulin (IgG) to ovalbumin modified by dopamine oxidation. The presence of antibody reactivity to quinone-modified proteins could contribute to or amplify the inflammatory response in PD.     

Correlation of desensitisation of platelet activating factor (PAF) receptors with intensity of inflammation and intestinal PAF content during experimental ileitis in guinea pig

The anti-phenolic glycolipid-I (PGL-I) assay as currently applied for leprosy is conceived as an early marker of asymptomatic infection, early disease diagnosis and cure monitoring. Its use as a prognostic marker of reaction is still a matter of controversy. We conducted a case-control study to investigate whether IgM and IgG anti-PGL-I antibodies could discriminate patients at increased risk of developing reactions. Eligible cases were untreated leprosy patients at the onset of type 1 and type 2 reactions recruited from among 600 concurrent, newly detected, untreated leprosy patients attending an outpatient clinic in central Brazil. For the patients with reaction, approximately the same number of leprosy cases without reaction matched as to bacterial index (BI), age and gender were randomly selected. Individuals without clinical leprosy were evaluated as healthy controls. Sera from type 1 reaction (N = 43) and type 2 reaction (N = 26) patients were tested by an ELISA using PGL-I synthetic disaccharide-BSA antigen and 1:300 sera dilution (cut-off point > or = 0.2 OD). Antibody profiles were evaluated by exploratory data analysis and reverse cumulative distribution curves. The IgG anti-PGL-I response did not have a defined pattern, being detected only at low levels. Our results indicate that leprosy patients, independently of their reactional status, produce high levels of IgM anti-PGL-I, demonstrating a strong correlation between the magnitude of antibody response and the BI. Patients with a higher BI were at least 3.4 times more prone to produce an antibody response compared to healthy controls.     

Ultraviolet-B irradiation of leukapheresis. Products: dose-response relationship with the mixed lymphocyte reaction

Our aim was to study the influence of infection with the respiratory syncytial virus (RSV) in non-hospitalized infants on sensitization to aeroallergens and the early manifestation of atopy. Six hundred and nine infants from the prospective German Multicenter Cohort Study on Atopy were included, 38% of whom had an elevated atopic risk. RSV IgG and IgM antibodies were tested by ELISA with gradient purified RSV antigen. Specific IgE against mites, cat dandruff, birch and grass pollens and relevant nutritional antigens were tested with CAP-RAST-FEIA (Pharmacia, Sweden). Of the cord sera 99% were positive for RSV-IgG, 44.7% at one year and 64.2% (n = 265) at two years of age. The positivity rate after 12 months varied with the season of birth, the number of siblings and the degree of exposure to tobacco smoke; and correlated closely with attacks of wheezing during infancy. Twenty (2.8%) children were found to be sensitized against at least one aeroallergen at one year, and 28 (10.5%) at two years. By the first birthday, mite sensitization (n = 3) could only be seen in the RSV-infected children; grass pollen sensitization (n = 9) was associated with RSV seropositivity (logistic regression model including the confounders mentioned above: with RSV IgG < p = 0.048 > and IgM < p = 0.0006 >), as was birch sensitization (n = 5) with RSV IgM (p = 0.009). No such differences could be detected at two years. No correlation of RSV seropositivity to any allergic manifestation could be found. We conclude, that it is only in the first year of life, that RSV infection plays a significant role in promoting sensitization against aeroallergens, which do not at this time produce allergic symptoms.     

Capillary haemangiomas in association with morning glory disc anomaly

The potential roles of specific antibodies of the different immunoglobulin G (IgG) subclasses in the serological diagnosis of cystic echinococcosis (CE) and alveolar echinococcosis (AE) were investigated by an enzyme-linked immunosorbent assay based on hydatid fluid as antigen. Specific antibodies of subclass 1 were found to be of major importance. In sera collected at the time of diagnosis (i.e., before any therapeutic intervention was initiated) they could be demonstrated in 14 of 15 sera from patients with CE and in all 12 sera from patients with AE. The most discriminatory and the most specific antibodies found in this study belonged to IgG subclass 4. Only one false-positive reaction was observed with 253 sera from healthy volunteers, and no cross-reactions occurred in 80 sera from patients with different parasitic infections. Specific IgG4 antibodies could be demonstrated in 61.0 to 66.7% (CE) or 47.6 to 66.7% (AE) of the cases. Antibody levels of IgG subclass 2 were elevated only moderately, and subclass 3 antibodies were detected in a few cases only. In addition, nonspecific reactions in sera of healthy volunteers or patients with other parasitic infections could partially be attributed to antibodies of subclasses 2 and 3.     

Antibodies to beta 2-glycoprotein-I: urea resistance, binding specificity, and association with thrombosis

The aim of the present study was to evaluate the urea resistance and binding characteristics of anti-beta 2-glycoprotein I (anti-beta 2GPI) antibodies using standard anticardiolipin (aCL) and anti-beta 2GPI enzyme immunosorbent assays (ELISAs). Sera from patients with antiphospholipid syndrome (APS) (n = 22) and non-APS (n = 24), positive in a standard aCL ELISA, were tested in an anti-beta 2GPI ELISA performed in polystyrene-irradiated ELISA plates. Urea resistance aCL and anti-beta 2GPI ELISAs were performed by measuring the ability of antibodies to recognize antigen in the presence of 2 M urea. The serum dilution after urea treatment (D) expressed as a percentage of the serum dilution without urea treatment (D(o)) corresponding to the same optical density was defined as residual activity (RA = 100 D/D(o)). The higher the RA, the higher the resistance of the antibodies to urea. APS compared to non-APS sera had higher aCL binding (absorbance values ranging between 0.180 and 1.400; median, 0.717 vs 0.120-1.273; median, 0.250, respectively; P < 0.004). Six APS patients' sera had low aCL levels but they expressed RA > or = 30%. Anti-beta 2GPI antibodies were detected in 15 of 22 APS vs 3 of 24 non-APS patients (P < 0.03); RA > or = 30% was detected in 15 of 22 APS vs 1 of 23 non-APS patients (P < 0.004). Using a CL affinity column, antibodies were purified from three APS anti-beta 2GPI negative and three non-APS anti-beta 2GPI-positive patients and tested in a aCL ELISA, using highly purified bovine serum albumin (BSA) as a blocking agent (modified ELISA); reactivity was not detected in two APS and one non-APS sera. On the contrary, the reactivity of the purified antibodies was high when beta 2GPI was incubated with CL in the ELISA plates; thus some anti-beta 2GPI negative sera from APS patients recognized the CL/beta 2GPI complex, rather than CL or beta 2GPI alone. In conclusion, anti-beta 2GPI antibodies are common in the APS patients, but a number of such patients recognize the CL/beta 2GPI complex and not CL or beta 2GPI. Antibodies to either beta 2GPI or the CL/beta 2GPI complex derived from APS sera present a high resistance to urea. Anti-beta 2GPI antibodies of low urea resistance exist in a minority of non-APS patients with autoimmune disease.     

Elevated levels of lung surfactant protein A in sera from patients with idiopathic pulmonary fibrosis and pulmonary alveolar proteinosis

An enzyme-linked immunosorbent assay using monoclonal antibodies to human lung surfactant protein A (SP-A) was applied to sera from patients with lung diseases. We examined whether SP-A appears in the sera of patients with diseases that are known to cause alterations in surfactant composition in bronchoalveolar lavage fluids, and we characterized the SP-A that was found. The level of SP-A in sera from 57 healthy volunteers was 45 +/- 3 ng/ml (mean +/- SEM). The levels in patients with idiopathic pulmonary fibrosis (IPF) (205 +/- 23 ng/ml, n = 32) and pulmonary alveolar proteinosis (PAP) (285 +/- 23 ng/ml, n = 6) were significantly higher than those in healthy control subjects (p < 0.01), whereas those of sarcoidosis (n = 16), pneumonia (n = 14), and tuberculosis (n = 14) were 52 +/- 27 ng/ml, 65 +/- 11 ng/ml, and 49 +/- 23 ng/ml, respectively. Electrophoresis and immunoblotting analysis demonstrated that the fraction isolated from serum of a patient with PAP or IPF by anti-SP-A immunoaffinity column chromatography consisted chiefly of human IgG and IgM, and that it also contained SP-A. Furthermore, IgG was found in preparation of purified human SP-A. SP-A was demonstrated to bind to nonimmune IgG coated onto microtiter wells. Gel filtration analysis revealed that serum SP-A was eluted at fractions of larger molecular size than was the purified SP-A. These findings suggest that SP-A appears in the bloodstream as a complex with immunoglobulin in IPF and in PAP.     

Cross-reactivity of anti-10kD heat shock protein antibodies in leprosy and tuberculosis patients

The response to recombinant 10-kD heat shock protein (HSP) of Mycobacterium leprae (rML10) was evaluated by indirect ELISA in sera from leprosy patients, household contacts, tuberculosis patients and healthy controls in a leprosy-endemic area in the North East of Argentina. Some technical parameters were analyzed: within-assay and between-assay variability, dose-response curves and detectability indexes (specificity and sensitivity) of ELISA applied to measure anti-10 kDa antibodies. High levels of these antibodies have already been reported in positive bacilloscopy patients; herein we have also demonstrated that tuberculosis patients sera cross-react with this M. leprae antigen. This test seems to have a low sensitivity and specificity for leprosy detection; it confirms that antibodies against highly conserved HSP antigens are important in the polyclonal response against mycobacterial epitopes in leprosy as well as in tuberculosis.     

Annexin V autoantibodies in rheumatoid arthritis

OBJECTIVE: To investigate the occurrence of anti-annexin V autoantibodies in sera of patients with rheumatoid arthritis to assess involvement with the disease and any relation to glucocorticoid treatment. METHODS: Anti-annexin V antibodies were measured by an enzyme linked immunosorbent assay (ELISA) which used the purified human recombinant protein as antigen. RESULTS: Concentrations of anti-annexin V autoantibodies, predominantly of the IgG class, were significantly raised in sera from patients with rheumatoid arthritis compared to normal controls. This was not correlated with other indices of disease activity such as erythrocyte sedimentation rate or C reactive protein and was unrelated to glucocorticoid treatment. CONCLUSIONS: Extracellular annexin V provides an antigenic stimulus for autoantibody production and its in vivo expression is independent of glucocorticoid control. Such autoantibodies may have a detrimental role in the arthritic condition by interfering with putative functions of annexin V, including collagen type II binding, inhibition of phospholipase A2 activity, and Fc receptor activity.     

A comparison of the characteristics of circulating anti-myeloperoxidase autoantibodies in vasculitis with those in non-vasculitic conditions

Although circulating anti-neutrophil cytoplasmic antibodies (ANCA) specific for myeloperoxidase (MPO) are strongly associated with the presence of vasculitis, they have been described in sera from patients with other conditions. High levels of anti-MPO antibodies can also persist in sera from patients with vasculitis despite the achievement of clinical remission. One possible interpretation is that a potentially pathogenic subset of anti-MPO antibodies exists, which is only present in patients with active vasculitis. We therefore compared the characteristics of anti-MPO antibodies in sera from patients with active vasculitis (n = 18) with those present in remission (n = 9) and in a disease control group (n = 10) without clinical evidence of vasculitis. The class, subclass and ability of anti-MPO antibodies from the three groups of patients to recognize three different conformational epitopes were analysed using ELISA-based techniques. The expression of an idiotope, designated 9G4, was also examined. Epitope recognition by anti-MPO antibodies from all patients tested was found to be similar. Sera from patients with active vasculitis showed an over-representation of IgG4 subclass anti-MPO antibodies and a more frequent presence of IgM class anti-MPO antibodies. In disease controls, IgG1 anti-MPO antibodies were predominant. In vitro, neutrophil activation by ANCA has been shown to be dependent on engagement of neutrophil FcgammaRIIa receptors following binding of these autoantibodies to surface-expressed ANCA antigens. We found that active vasculitis may be associated with the presence of circulating anti-MPO antibodies which do not significantly bind this receptor, suggesting that mechanisms other than those dependent on FcgammaRIIa binding should be explored. In addition, the expression of the 9G4 idiotope on anti-MPO antibodies in 60% (12/18) of patients with active vasculitis and 20% (2/10) of disease control patients may indicate a common origin for anti-MPO antibodies in different individuals.     

Rebox effect on exercise-induced acute inflammation in human muscle

BACKGROUND: The first encounters with allergens seem to influence the development of allergy. Food antigens have been detected in sera as free antigens and in complexes with IgG but less is known about the presence of inhalant allergens. OBJECTIVE: To investigate the presence of the major cat allergen Fel d 1, either as free allergen and/or in complexes with IgG and IgE antibodies in sera from atopic children. METHODS: Serum samples from 33 cat allergic asthmatic children, 7-17 years old, and 15 non-allergic controls were investigated for the presence of Fel d 1 by ELISA (detection limit 0. 13 microg/L). To detect immune complexes (IC), the IgG fraction from Fel d 1 positive sera was purified by affinity chromatography. Purified and non-absorbed material was then analysed for allergen content and specific IgG antibody levels. Immune complexes with Fel d 1 IgE were detected by coupling anti-Fel d 1 MoAb to paramagnetic particles. RESULTS: Fel d 1 was detected (0.15-1.8 microg/L) in 23 of the 33 patients (70%) but not from any of the controls. Eighteen samples contained IgE-Fel d 1 IC and two of four tested samples contained Fel d 1 in the IgG fraction. Electrophoresis and Western blotting of IgG purified material using anti-Fel d 1 MoAb corroborated the presence of IgG-Fel d 1 IC. CONCLUSION: Free-circulating inhalant allergen and IC with allergens may contribute to maintaining immune responsiveness and sensitivity.     

Increased immunoglobulin binding to cerebral endothelium in patients with antiphospholipid antibodies

BACKGROUND AND PURPOSE: There is a strong link between antiphospholipid antibodies and stroke. The mechanism of action of antiphospholipid antibodies is unknown. Most theories of pathogenesis center around platelet or endothelial cell dysfunction. Our aim was to determine if there were immunoglobulins in the sera of patients with antiphospholipid antibodies that bind human brain microvascular endothelial cells. METHODS: We studied sera from three groups of subjects: patients with antiphospholipid antibodies and stroke (group 1), healthy control subjects (group 2), and patients with stroke but without antiphospholipid antibodies (group 3). We isolated human brain microvascular endothelial cells from temporal lobectomy specimens and used a cellular enzyme-linked immunosorbent assay (ELISA) to measure immunoglobulin binding to endothelial cells derived from human brain and from human umbilical vein. We used a chromium release assay to measure cytotoxicity. RESULTS: Patients with antiphospholipid antibodies and stroke had significantly higher immunoglobulin binding to human brain microvascular endothelial cells than subjects in the other groups ([ELISA index+standard deviation], 63 +/- 37 [group 1] versus 7 +/- 7 [group 2] versus 7 +/- 7 [group 3], P < .001). There was, however, poor correlation between binding to brain endothelial cells and binding to cardiolipin. The binding to brain microvascular cells was not specific to brain endothelium, as similar results were found in an ELISA using human umbilical vein cells. There was no evidence of complement-mediated brain endothelial cell cytotoxicity. CONCLUSIONS: Patients with stroke and antiphospholipid antibodies frequently have human brain microvascular endothelial-reactive antibodies in their serum. These antibodies are distinct from those to cardiolipin. We found no evidence that these antibodies are cytotoxic.     

The effects of interferon-gamma on the central nervous system

A 165bp DNA fragment derived from the 12 kDa subunit of Echinococcus granulosus antigen B (AgB), a major hydatid cyst fluid antigen was cloned in the pMa1-c2 expression vector. A 52 kDa maltose binding-AgB fusion protein (rAgB.MBP) was produced and inclusion bodies containing the fusion protein were solubilised in urea and affinity purified on an amylose-Sepharose 6B column. The immunogenicity of the purified recombinant antigen for IgG4 antibody detection was tested with human serum using immunoblotting, ELISA and dot-ELISA assays and compared to native AgB. Both recombinant and native AgB preparations were highly reactive for human IgG4 antibodies in serum of cystic echinococcus (CE) patients. Recombinant AgB.MBP (rAgB.MBP) showed approximately 65% sensitivity in detection of IgG4 serum antibodies by ELISA from confirmed CE patients. Cross-reactivity (33%) occurred with alveolar echinococcosis (E. multilocularis) sera but recombinant AgB showed no seroreactivity with sera from other helminth infections tested (schistosomsis, onchocercsis, cysticercosis) or from uninfected individuals residing in CE endemic or non-endemic regions. The serologic sensitivity (63%) for IgG4 antibodies of a native AgB fraction enriched from human hydatid cyst fluid was similar to that for recombinant AgB (65%) though specificity was slightly lower (81%). A dot-ELISA for detection of total IgG, incorporating the rAgB.MBP resulted in 74% sensitivity and 88% specificity for human CE and 93% sensitivity and 65% specificity for native AgB. Recombinant AgB is a potential replacement for native antigens currently being used and could provide a better standardised E. granulosus specific test for clinical confirmation for CE especially for IgG4 antibody detection which appears to be predominantly associated with advanced disease.     

Association of interleukin 6 release from endothelial cells and pulmonary hypertension in SLE

OBJECTIVE: To investigate how sera from 37 patients with systemic lupus erythematosus (SLE) stimulate interleukin (IL) 6 release from IL-1beta pretreated endothelial cells and compare these effects to those of sera from 16 normal controls. METHODS: Endothelial cells pretreated 18 h with IL-1beta (5 U/ml) were incubated 2 h with sera diluted 10-fold with phosphate buffered saline (PBS). IL-6 concentrations in endothelial culture supernatants collected after incubation were measured by ELISA. RESULTS: Compared with PBS, sera from controls and 24 patients with SLE suppressed IL-6 release from IL-1beta pretreated cells. However, sera from 13 patients with SLE augmented IL-6 release. Of note, sera from 5 patients with pulmonary hypertension induced the highest level of IL-6 release. IgG from control sera suppressed IL-6 release, whereas F(ab')2 did not. Both IgG and F(ab')2 from the sera of patients with SLE with pulmonary hypertension augmented IL-6 release from IL-1beta pretreated cells. CONCLUSION: IgG antiendothelial cell antibodies from patients with SLE may be associated with the pathogenesis of SLE and pulmonary hypertension.     

Specific IgA and IgG antibodies to house dust mite Dermatophagoides farinae in nasal secretions

As far as we know, IgA and IgG antibodies to purified house dust mite allergens Der fI and Der fII in nasal secretions have never been documented. Therefore, we determined specific IgA, SIgA and IgG antibodies (abs) to crude extract of D. farinae and its purified allergens Der fI and der f II in nasal secretions collected by aspiration from 34 normal subjects, 25 untreated nasal allergic patients and 28 treated nasal allergic patients on parenteral immunotherapy by means of an avidin-biotin ELISA. The following results were obtained. (1) The specific IgA, SIgA and IgG abs to each of the three kinds of allergens correlated with each other. The groups of patients with nasal allergy (both treated and untreated) showed higher levels of specific IgA, SIgA and IgG abs to the allergens than the normal group. (2) In the group of treated patients, the levels of specific abs were not correlated with the clinical improvement of symptoms or the degree of response to nasal challenge. (3) The treated patients failed to show significantly higher levels of abs in nasal secretions than the untreated patients. (4) The specific IgA and SIgA abs in nasal secretions seemed to be predominantly produced locally, and IgG abs might be transudated from the circulation.     

Autoantibodies to M2 mitochondrial autoantigens in normal human sera by immunofluorescence and novel assays

Primary biliary cirrhosis (PBC) is characterized by the presence of antimitochondrial antibodies (anti-M2), directed against the E2 subunits of the 2-oxo-acid dehydrogenase complexes (2-OADC), chiefly pyruvate dehydrogenase complex (PDC-E2). We present here a detailed study, based on a large panel of normal sera, of the specificity of tests for anti-M2 by immunofluorescence and for anti-PDC by other assays for the diagnosis of PBC. The assays for anti-PDC included immunoblotting with bovine heart mitochondria, ELISA using recombinant PDC-E2 and an enzyme inhibition assay using purified porcine PDC. The positivity rates for normal sera were 0 (0/170), 2 (4/201), 1.5 (3/198) and 0% (0/186) for immunofluorescence, immunoblotting, ELISA and the enzyme inhibition assay, respectively. The seven positive reactions detected either by immunoblotting (n = 4) or ELISA (n = 3) were negative by the other three assays and in no instance did biochemical indices give any indication of chronic liver disease. Thus, as judged by reactivity with normal sera, the specificity of a positive test for the antibody to the major M2 autoantigen (PDC-E2) is 100% for immunofluorescence and the enzyme inhibition assay, 98% for immunoblotting and 98.5% for ELISA.     

Antibody isotypes, including IgG subclasses, in Ecuadorian patients with pulmonary paragonimiasis

An ELISA test was developed to detect Paragonimus-specific antibodies, including IgG subclasses, using P. mexicanus crude water-soluble antigens. The test was standardized to detect antibodies in sera of Ecuadorian patients with pulmonary paragonimiasis and negative controls from the endemic area. The detected mean levels of IgG (0.753, SEM: 0.074) and IgM (0.303, SEM: 0.033) were significantly elevated (P < 0.05). Within the IgG subclasses, IgG4 showed the highest detected mean level (0.365, SEM: 0.116) and the other three subclasses showed considerably lower mean levels (IgG1, 0.186 SEM: 0.06; IgG2, 0.046 SEM: 0.01; IgG3, 0.123 SEM: 0.047). The number of P. mexicanus eggs found in sputum of infected individuals showed a positive correlation with the level of antibodies detected for IgM, IgG and its subclasses (P < 0.001). The relevance of these findings in Ecuadorian patients suffering from pulmonary paragonimiasis is discussed.