Characterization of an Uncinocarpus reesii-expressed recombinant tube precipitin antigen of Coccidioides posadasii for serodiagnosis.

Early and accurate diagnosis of coccidioidomycosis, also known as Valley fever, is critical for appropriate disease treatment and management. Current serodiagnosis is based on the detection of patient serum antibodies that react with tube precipitin (TP) and complement fixation (CF) antigens of Coccidioides. IgM is the first class of antibodies produced by hosts in response to coccidioidal insults. The highly glycosylated ß-glucosidase 2 (BGL2) is a major active component of the TP antigen that stimulates IgM antibody responses during early Coccidioides infection. The predominant IgM epitope on BGL2 is a unique 3-O-methyl-mannose moiety that is not produced by commonly used protein expression systems. We genetically engineered and expressed a recombinant BGL2 (rBGL2ur), derived from Coccidioides, in non-pathogenic Uncinocarpus reesii, a fungus phylogenetically related to the Coccidioides pathogen. The rBGL2ur protein was purified from the culture medium of transformed U. reesii by nickel affinity chromatography, and the presence of 3-O-methyl mannose was demonstrated by gas chromatography. Seroreactivity of the purified rBGL2ur protein was tested by enzyme-linked immunosorbent assays using sera from 90 patients with coccidioidomycosis and 134 control individuals. The sensitivity and specificity of the assay with rBGL2ur were 78.8% and 87.3%, respectively. These results were comparable to those obtained using a proprietary MiraVista Diagnostic (MVD) IgM (63.3% sensitivity; 96.3% specificity), but significantly better than the ID-TP assay using non-concentrated patient sera (33.3% sensitivity; 100% specificity). Expression of rBGL2ur in U. reesii retains its antigenicity for coccidioidomycosis serodiagnosis and greatly reduces biosafety concerns for antigen production, as Coccidioides spp. are biological safety level 3 agents.

Concordance between Aspergillus-specific precipitating antibody and IgG in allergic bronchopulmonary aspergillosis.

BACKGROUND: Several serological tests for specific precipitin or IgG are available to demonstrate type III hypersensitivity reactions to Aspergillus species and are essential for infectious fungal disease diagnosis. These assays are also important for allergic bronchopulmonary aspergillosis (ABPA) diagnosis; however, their concordance in ABPA was not well studied. METHODS: Fifty-two ABPA patients diagnosed based on ISHAM criteria were enrolled. Precipitins and IgG specific to Aspergillus fumigatus, Aspergillus niger, Aspergillus flavus, or Aspergillus terreus were measured using Ouchterlony double immunodiffusion tests and ImmunoCAP method, respectively. A. fumigatus-specific IgG was also determined using complement-fixation (CF) method. RESULTS: Forty-eight percent of cases were double-positive for A. fumigatus-specific precipitin and IgG (ImmunoCAP), whereas 3 (6%) and 14 (28%) cases were positive for precipitin or IgG alone, respectively. Kappa coefficient between these measurements was 0.32, suggesting poor concordance. Double-positive cases were more likely to present: Aspergillus sp. in sputum culture, lower pulmonary functions, peripheral blood eosinophilia, higher total IgE and A. fumigatus-specific IgG titer than precipitin-negative cases. A. fumigatus-specific IgG (CF) was positive only in 8 (15%) cases. The presence of A. fumigatus-specific precipitin or IgG was associated with antibodies specific for other Aspergillus spp., suggesting cross-reactivity. CONCLUSIONS: Positive rate of A. fumigatus-specific precipitin or IgG (ImmunoCAP) was superior to IgG (CF), but relatively poor concordance was noted between precipitin and IgG (ImmunoCAP). Positive precipitin for A. fumigatus suggests more active diseases. Cross-reactivity may exist between antibodies to different Aspergillus spp. Therefore, the type III hypersensitivity results in ABPA diagnosis should be carefully evaluated.

Immune ring formation associated with cytomegalovirus endotheliitis.

PURPOSE: To describe immune ring (IR) formation as a manifestation of cytomegalovirus (CMV) endotheliitis. DESIGN: Retrospective observational case series. METHODS: Review of all consecutive CMV anterior uveitis and endotheliitis patients seen at the Singapore National Eye Centre for the occurrence of an IR. CMV infection was diagnosed by polymerase chain reaction analysis of the aqueous. RESULTS: None of the 72 eyes with CMV anterior uveitis had an IR formation. Four episodes of IR formation were seen in 3 eyes (14.3%) of 21 cases of CMV-positive endotheliitis. All were unilateral and all 3 patients were human immunodeficiency virus negative middle-aged Chinese males. The IR developed 2 to 7 months after occurrence of the endotheliitis and the aqueous was positive for CMV during 2 of the episodes of IR formation. In 2 eyes, the IR occurred at the completion of a course of systemic ganciclovir when the aqueous was negative for CMV. The immune rings resolved with combination therapy of topical prednisolone acetate 0.12% and ganciclovir. Patient 1 had a recurrence of the IR 4 months after stopping treatment but again resolved following treatment with ganciclovir and topical corticosteroids. CONCLUSION: Corneal immune rings can occur as a result of CMV infection. Hence CMV infection may have to be considered in such cases.

Severe asthma with fungal sensitization: a case report and review of literature.

There is a substantial body of evidence supporting an association between asthma severity and fungal exposure and sensitization. Fungal allergens are a recognized risk factor for severe asthma. We describe the case of a 44-year-old asthmatic whose asthma control deteriorated after moving to a new flat with walls covered in mould. Allergic bronchopulmonary aspergillosis was excluded. Although sensitization to Candida was demonstrated by a positive Candida-specific radioallergosorbent test, the patient did not entirely satisfy the criteria for a diagnosis of allergic bronchopulmonary candidiasis. The patient's asthma control improved after engaging in a monthly washing regimen of the walls. This case further demonstrates the association between fungal sensitization and asthma severity. The term severe asthma with fungal sensitization has been recently coined to describe this phenomenon.

Theoretical and experimental investigation of immunoprecipitation pattern formation in gel medium.

We present the results of our comprehensive study of precipitation pattern formation by interacting immunogenic proteins in a gel medium. Formation of immunoprecipitation patterns was studied both theoretically and experimentally. Based on a system of reaction-diffusion equations, continuous deterministic description provides a quantitative model of reaction kinetics. Discrete stochastic microscopic description was used to supplement the results of reaction-diffusion model by mimicking product aggregation that contributes to a deeper understanding of the mechanism that governs the phenomenon. Our studies have shown that the mechanism of immunoprecipitation pattern formation is specific for protein precipitation and differs from such mechanisms for any inorganic or biological substances. By microscopic examination, we demonstrated that immunoprecipitation patterns can have a microstructure. We found that the microscopic structure of immunoprecipitation patterns results from multicomponent composition of antiserum.

Health effects of exposure to organic dust in workers of a modern hatchery.

The aim of the presented study was to determine the health status of workers occupationally exposed to moderate amounts of organic dust, employed in a modern hatchery with an efficient ventilation system. A group of 32 hatchery workers was examined. As a reference group, 50 urban dwellers not exposed to any kind of organic dust were examined. All people were interviewed for the presence of work-related symptoms and subjected to physical and spirometric examinations. Blood sera were examined for the presence of precipitins against 13 antigens associated with organic dust, and for the presence of total and chicken-specific No significant differences were found between the spirometric values in the group of hatchery workers and the reference group. Positive precipitin reactions were noted mostly with the antigens of Gram-negative bacteria associated with organic dust. The frequencies of positive reactions to antigens of Escherichia coli and Acinetobacter baumannii in hatchery workers were significantly greater compared to the reference group (p<0.05). Precipitin reactions to Gram-positive non-branching bacteria, actinomycetes, fungi and birdactions to antigens of Escherichia coli and Acinetobacter baumannii in hatchery workers were significantly greater compared to the reference group (p<0.05). Precipitin reactions to Gram-positive non-branching bacteria, actinomycetes, fungi and bird protein, were rare or absent. The mean concentration of total IgE in sera of hatchery workers was nearly 3 times greater compared to the reference group, and the difference proved to be statistically significant (p<0.05). No specific IgE antibodies against chicken feathers were detected in the blood of hatchery workers and referents. In conclusion, the examined hatchery workers showed a moderate frequency of work-related symptoms, no decline in lung function and low reactivity to most microbial and bird protein allergens. These results suggest that the effects of exposure to organic dust in workers of modern hatcheries with an efficient ventilation system are less compared to the workers of poultry farms, such as broiler or egg laying houses.

Human sera with precipitating antibodies to human soluble immune complexes. A brief communication.

INTRODUCTION: Previous numerous papers by the senior author dealt with the human serum factor referred to as anti-antibody which is specifically directed against IgG antibodies that underwent molecular transformation in the course of the reactions with their corresponding antigens. The reactions of this serum factor could be conveniently detected by means of agglutination of Rh-positive erythrocytes sensitized by anti Rh antibodies. No precipitation tests could be developed. MATERIAL AND METHODS: Most studies were conducted by means of double diffusion in gel precipitation. RESULTS: A rheumatoid arthritis serum, G, was noted that produced a strong reaction of double diffusion in gel precipitation with serum samples of a renal graft recipient, T. Further screening detected one more rheumatoid arthritis serum reacting with T; of 28 sera from renal graft recipients, 6 reacted in a similar way to T, but the reactions were considerably weaker and poorly reproducible. Evidence was presented that the precipitin in the two rheumatoid arthritis sera under study had properties of previously described anti-antibody. CONCLUSIONS: Sera with precipitating anti-antibodies may serve as exquisite reagents for detection of soluble immune complexes in human sera.

Significance of precipitins and asymptomatic lymphocytic alveolitis: a 20-yr follow-up.

What is the significance of serum precipitins and lymphocytic alveolitis in a healthy subject exposed to antigens responsible for hypersensitivity pneumonitis (HP)? This study was done to evaluate the 20-yr outcome of asymptomatic dairy farmers with or without precipitins or lymphocytic alveolitis in their bronchoalveolar lavage (BAL). Twenty-seven of the initial 43 farmers were restudied. Of the missing 16, 11 refused the follow-up, three had died and two could not be located. The restudied farmers had a clinical evaluation, lung function tests and a high resolution computed tomography (HRCT). Only one subject, an asthmatic, had dyspnoea, none described present or past symptoms suggestive of HP. Of those who were no longer on the farm, none had quit because of respiratory problems. Lung function changes were as expected with aging. The only difference was that farmers with positive precipitins had a statistically significant greater decrease in their forced expiratory volume in one second than the sero-negative farmers. Four HRCTs showed signs of minimal parenchymal abnormalities; these were not more prevalent for subjects with or without precipitins or BAL lymphocytosis. Serum precipitins and asymptomatic lymphocytic alveolitis in an asymptomatic, exposed dairy farmer have no clinically meaningful long-term consequences.

Deficiency of complement-dependent prevention of immune precipitation in systemic sclerosis.

BACKGROUND: Previous studies have indicated that complement may be activated or inherently abnormal in systemic sclerosis (SSc), and it has been suggested that immune complex deposition plays a part in the microvascular damage of this disease. OBJECTIVE: To study several aspects of the complement system in 24 patients with SSc. METHODS: Complement dependent prevention of immune precipitation (PIP) was measured by a sensitive enzyme immunoassay, levels of C1q, C4, and C3 by rocket immunoelectrophoresis, C4 allotypes by high voltage agarose electrophoresis, and C4A, C4B, and C3d by an enzyme linked immunosorbent assay (ELISA). RESULTS: PIP was markedly decreased in the patients with SSc (p<0.001). Abnormal complement activation was detected in nine patients as raised levels of the complement split product C3d. However, a relation between low PIP and complement activation was not seen. PIP was significantly lower in patients who carried the C4A*Q0 allotype (p=0.03), and a strong correlation was found between PIP and C4A concentration (p<0.00001). The PIP defect may, at least in some patients, be associated with the initial phase of the disease. CONCLUSIONS: The results show a previously unrecognised functional defect of complement in SSc; the defect correlates with low levels of classical pathway components and, in particular, C4A.

Partial characterization of serum immunoglobulins of Oreochromis mossambicus.

Serum immunoglobulins of O. mossambicus were purified using chromatography methods--CM affinity gel blue chromatography followed by two step purification involving a combination of ion-exchange and gel filtration chromatography. Studies revealed that O. mossambicus produces only one class of high molecular weight macroglobulin as determined by molecular sieving by Sepharose CL 6-B. Immunoelectrophoresis of purified O. mossambicus serum against rabbit anti O. mossambicus serum gave only a single precipitin line. Further analysis of the immunoglobulin by SDS-PAGE showed that the IgM macroglobulin weighs about 900,000 Da, composed of mu-like heavy chain weighing about 90 kDa each and light chains weighing about 30 kDa each.

[Autoimmune processes and biocompatibility in local damage of endothelium of the heart and its vessels]. / Autoimunni protsesy ta biosumisnist' pry vohnyshchevykh urazhenniakh endoteliiu sertsia ta ioho sudyn.

At immunoproductive processes in endothelium of patients with coronary atherosclerosis and endocarditis a phase dynamics of innate immunity represented by polytypic Ca(2+)-dependent autoprecipitating proteins of nonclonal recognition of surface cell membrane components (CMC) have been revealed. In case of coronary atherosclerosis the organism reaction to expressed CMC is manifested basically by intensified synthesis of cathodic complement-C3-similar autoprecipitin, but in case of endocarditis--by consumption of one with formation of anodic autoprecipitin and by intensified synthesis of autoprecipitating immunoglobulin G. These membranotropic autoimmune reactions are identical with the organism reactions to alien (heterologous) agents and differentially participate in development both of basic disease and perioperational complications after cardiac operations using trans- and implants, hence, they should be taken into account during membrane-stabilising and immunocorrecting therapy of recipients.

[Anti-pseudomonas-specific precipitins as a marker of chronic infection in patients with cystic fibrosis]. / Le precipitine specifiche antipseudomonas come marker di infezione cronica nel paziente fibrocistico.

AIMS: The authors underline the characteristics of Pseudomonas aeruginosa and its methods of action on bronchial mucosa in cystic fibrosis. They then discuss the two concepts of "colonisation" and "chronic infection". METHODS: The level of "infection" was evaluated using an immunoelectrophoretic method involving the precipitation of bands of specific precipitins. A technical description of the method is included. The authors illustrate the use of the method in 78 cases of cystic fibrosis, comparing the positive results obtained using precipitin electrophoresis with the results of direct bacteriological findings. RESULTS: Using the bacteriological criteria, a total of 26.9% of patients were diagnosed as infected, whereas this percentage rose to 32.1% using precipitins. 87.2% of cases were concordant using both methods. CONCLUSIONS: As a practical solution, the authors recommend that the two methods are combined, thus obtaining a marked reduction in the number of false positives with obvious consequences in terms of therapeutic decisions.

[Two cases of acute eosinophilic pneumonia with precipitating antibody against Trichosporon cutaneum and Trichoderma viride].

Two cases of acute eosinophilic pneumonia are described. The patients presented with an acute febrile illness, dry cough, severe hypoxemia and diffuse pulmonary infiltrates. Total cell count and the number of eosinophils were increased in bronchoalveolar lavage fluid. The TBLB specimen showed eosinophilic infiltration of alveolar walls and spaces. Precipitating antibodies against Trichosporon cutaneum and Trichoderma viride were noted in the patients' sera, and environmental provocation tests gave positive results. The clinical features of acute eosinophilic pneumonia resemble those of summer type hypersensitivity pneumonitis. From these results, we consider that there is a certain degree of overlap between the two diseases.

[Detection of circulating antibodies in the blood and immune complex precipitates of patients with autoimmune diseases (preliminary report)]. / Keringö autoantitestek vizsgálata autoimmun betegségben szenvedök szérumában és immunkomplex precipitátumaiban (Elözetes közlemény).

Antibodies against ENA (Extractable Nuclear Antigens), SSA/Ro and SSB/La were investigated in parallel in the serum and the immune complex precipitates of 41 patients with autoimmune disease (10 systemic lupus erythematosus, 23 discoid lupus erythematosus, 3 subacute cutaneous lupus erythematosus, and 5 progressive systemic sclerosis). It was demonstrated that in some cases the autoantibody spectrum of the serum did not coincide with that of the immune complex precipitate. In the majority of patients the protein content of isolated immune complexes was increased and the increase was attributed to IgG solely. In some patients antibodies appeared not only in the serum but also in immune complex, even in 4 patients (2 systemic lupus erythematosus, 1 subacute cutaneous lupus erythematosus, and 1 progressive systemic sclerosis) antibodies were detected only in the immune complex precipitate.

The long-term evolution of immune deposits in passive Heymann nephritis.

1. To study the long term course of passive Heymann nephritis (PHN), 42 adult male Wistar rats were injected with rabbit anti-FX1A serum (PHN group) and 42 rats received normal rabbit serum (control group). Two animals from each group were sacrificed 2 weeks after the inoculation and 10 animals each from the control and PHN groups were sacrificed 4, 13, 25 and 53 weeks later. 2. The PHN group exhibited a significant elevation in 20-h proteinuria which lasted from the first week (control group, 9.19 +/- 0.87; PHN group, 25.3 +/- 2.66) to the 25th week (control group, 22.6 +/- 2.15; PHN group, 66.7 +/- 10.4) except for week 17. From week 29 to week 53 there was no statistical difference between the 2 groups. 3. Light microscopy showed no difference between the kidneys of PHN and control rats. Immunofluorescence microscopy in PHN rats showed granular deposition of autologous and heterologous IgG on the glomerular basement membrane (GBM), whose intensity and pattern did not change during 53 weeks of observation. 4. When examined by electron microscopy the glomeruli of PHN rats showed: a) electron-dense deposits which were initially subepithelial and homogeneous and later intramembranous, granular and often surrounded by an electron-transparent halo; b) focal thickening of the GBM at the sites of intramembranous deposits; c) effacement of podocytes located close to the deposits; d) "penetration" of the podocytes into the GBM associated with the deposits; e) presence of osmiophilic granules in the cytoplasm of the podocytes located inside the GBM similar to the granules of the deposits next to them. The association of the penetration of the podocytes into the GBM with the deposits and the presence of the osmiophilic granules inside the foot process have not been described previously in PHN.

Formation of subepithelial dense deposits in rats induced by a monoclonal antibody against the glomerular cell surface antigen.

We developed a monoclonal antibody, H5H3, of IgG1 subclass by hybridization technique using spleen cells of mice immunized with plasma membrane fraction of isolated rat glomeruli. H5H3 recognized main bands at about 220 kD by immuno-overlay technique and bound to the glomerulus as well as brush border of proximal tubules by indirect immunofluorescence (IF) microscopy on normal rat kidney frozen sections. By immunoelectron microscopy (IEM) it bound to the surface of mainly glomerular epithelial cell and weakly to the endothelial cell. After injection to Wistar rats it remained granularly in the glomerulus for more than 2 weeks seen by IF. When rats were preimmunized with murine IgG 4 days before the injection of H5H3, mouse IgG, rat IgG and C3 were strongly visible granularly in the glomerulus in 14 days by IF. Numerous dense deposits were formed at subepithelial area seen by transmission electron microscopy. Perfusion experiment of H5H3 into rat left kidney showed granular distribution of mouse IgG in 48 h, indicating that the reaction occurred in situ. H5H3 bound diffusely in fine granular pattern on the surface of cultured glomerular epithelial cells (GEC) studied by IF and IEM. Antigenic redistribution occurred on GEC after incubation of H5H3 at 37 C. These results suggested the required conditions to form subepithelial immune dense deposits, namely that H5H3 after reaction with antigen could stay for long time in the glomerulus; that H5H3 became an antigen in autologous phase to induce large immune complexes; and H5H3 could induce antigenic modulation.

Precipitating antibodies against pigeon bloom antigens as a specific and sensitive measure of pigeon exposure.

Precipitating antibodies against pigeon bloom antigen and pigeon serum were evaluated as a measure of pigeon exposure in 62 male pigeon breeders compared with 40 unexposed control subjects. We found a combination of highly antigenic bloom extract and a sensitive counter current immunoelectrophoresis suitable for clinical estimation of pigeon exposure. The sensitivity of the test was 86% in never-smokers and 73% in smokers at the 98% specificity level. Smoking significantly reduced the humoral immune response to both antigens.

The clinical significance of autoantibodies to soluble cellular antigens in systemic lupus erythematosus.

The relationships between autoantibodies to soluble cellular antigens and clinical features in systemic lupus erythematosus (SLE) were investigated in a large clinical-serological study. The absence of these precipitins in serum was associated with a low prevalence of vasculitis and membranous nephropathy (MGN). Other significant findings were the associations between nRNP antibody and Raynaud's phenomenon and MGN, SSB antibody and sicca complex, PCNA antibody and a young age at onset, and Bu antibody and an old age at onset. However, the most impressive findings were in DA1-positive patients which showed a unique prevalence of photosensitive skin lesions, lymphoadenopathy and hepatosplenomegaly. The present study confirms the usefulness of antibodies to soluble cellular antigens in the classification of patients with SLE.

Complement-mediated inhibition of immune precipitation in rheumatoid vasculitis.

Complement-mediated inhibition of immune precipitation (CMIP) was measured in patients with rheumatoid arthritis (RA), rheumatoid vasculitis (RA VASC), patients with skin vasculitis not associated with a systemic connective tissue disease and normal healthy controls. CMIP was impaired in 100% (14/14) of the RA vasculitic patients, 60% (12/20) of the RA patients and 22% (2/9) of the dermovasculitic patients. The degree of impairment of CMIP was significantly greater in the RA vasculitic patients compared to the non-vasculitic patients. This difference was due to the significantly lower complement levels and the presence of higher concentrations of an inhibitor of CMIP in the RA vasculitic sera. The levels of this inhibitory activity correlated significantly with IgM rheumatoid factor concentration. Serial studies in three patients with RA vasculitis treated with corticosteroids and immunosuppressive drugs showed significant clinical improvement in two patients, which was associated with improvement in CMIP, reduction in circulating immune complex levels and reduction in IgM rheumatoid factor concentrations.