Microassay analyses of protein glycosylation.

To accurately characterize the carbohydrate moieties of oligosaccharide chains in glycosylated proteins, it is necessary to distinguish exactly which types of oligosaccharides are present at which site. We describe lectin overlay assays, which take advantage of the ability of lectins to distinguish between different types of glycoproteins via recognition of terminal sugars, thus allowing the chain type and peripheral antigenic components to be determined. Three microassays involving lectins are reported in this paper: non-protease-treated intact glycoproteins; glycopeptides released by prior digestion of the glycoprotein and then separated by HPLC; and release of sugars from glycoproteins by hydrazinolysis and then coupling them to a multivalent support.

The isolation and isoenzyme characterization of Leishmania braziliensis subsp. from patients with cutaneous leishmaniasis acquired in Belize.

Leishmanial organisms were cultivated from cutaneous lesions of British military personnel returning from Belize. Isoenzyme profiles of the freshly isolated organisms and 'marker' strains of New World Leishmania spp. were compared using 10 enzymes (ALAT, ASAT, ME, GPI, MPI, PGM, SOD, 6-PGDH, G-6-PDH and MDH), by starch gel electrophoresis. 19 of the 22 new isolates from Belize were isoenzymically indistinguishable from Leishmania braziliensis braziliensis (10 out of 10 enzymes) and clearly differentiated from L. b. guyanensis and L. b. panamensis (different in 6 out of 10 enzymes) and from L. mexicana mexicana and L. m. amazonensis (9 out of 10 enzymes). Two isolates closely resembled L. m. mexicana and one could not be positively identified. This is the first report of autochthonous human leishmaniasis caused by L. braziliensis group organisms as far north as latitude 16 degrees N.

Leishmania infecting man and wild animals in Saudi Arabia. 1. General survey.

Using up to 13 enzymes, biochemical characterization of 75 isolates of Leishmania made from man, wild animals and sandflies from a wide variety of localities in the Kingdom of Saudi Arabia has revealed the presence of L. major (two similar zymodemes), L. tropica (two zymodemes) and a parasite of the L. donovani-L. infantum complex. Zymodeme LON-4 of L. major has been found in 52 of 53 isolates so far characterized from man, from one specimen of Phlebotomus papatasi, from 15 Psammomys obesus, from one Meriones libycus and from one dog. One isolate from man has been identified as a new variant of L. major. This variant, zymodeme LON-65, varies from zymodeme LON-4 in a single enzyme. While this is the only example of zymodeme LON-65 identified so far, zymodeme LON-4 has also been obtained from Kuwait and Iraq. These are the first reports of L. major in Meriones libycus from Saudi Arabia and the first proven isolate from the dog in any country. L. tropica was identified from only two foci, whereas L. major appears to be widely distributed in the Kingdom. Two infants with kala-azar were found to be infected with a parasite apparently identical to zymodeme LON-42 of L. donovani (sensu lato) which also occurs in the highlands of Ethiopia.

Analysis of pigeon intestinal mucin allergens using a novel dot blot assay.

Many glycoproteins contain multiple glycosylation sites that can present multi-valent epitopes for antigenic recognition. Release of the oligosaccharides results in loss of avidity of antibody binding, which has been overcome by reforming clustered ligands, usually by reductive amination of free reducing oligosaccharides to poly-amine groups. We have adapted this approach to hydrazinolytic release of O-linked chains of mucin glycoproteins and 'one-pot' microscale coupling to poly-L-lysine (PLL). The conjugated PLL adheres to nitrocellulose membranes through washing procedures required for antibody or lectin overlay and detection. We show evidence for the applicability of this technique using lectin and antibody reactivity to the oligosaccharides of pigeon intestinal mucins, which have been implicated in the allergic disease pigeon fanciers' lung.

Isolation and characterization of three subpopulations of IgG in the common cat (Felis catus).

Although the cat is an important model for a number of human diseases little is known of its basic humoral immunity. In this study we have isolated three subpopulations of IgG from cat serum by DEAE anion-exchange chromatography and protein A affinity chromatography. Subpopulation 3 did not bind to DEAE equilibrated in 2 mM phosphate buffer pH 8.0 while subpopulations 1 and 2 were eluted, as a mixed population, with a salt gradient between 3 mM and 25 mM. When this fraction was loaded onto protein A-Sepharose two distinct peaks were always generated in a pH gradient (pH 8.0-2.1). The first of these, subpopulation 2, was eluted on average at pH 4.3 while the second, subpopulation 1, was eluted on average at pH 3.4. Subpopulations 1 and 2 had similar immunoelectrophoretic mobilities and isoelectric points while subpopulation 3 was more cathodic in both tests. Antisera produced in both mice and rabbits were rendered specific, by absorption of cross-reacting antibodies, for determinants on the heavy chain of each subpopulation as shown by Western blotting. The data suggest, but are not conclusive, that each of these subpopulations are distinct subclasses of IgG.

IgE responses in cats infected with Brugia pahangi.

Passive cutaneous anaphylaxis tests were used to examine the IgE responses of cats repeatedly infected with the filarial nematode Brugia pahangi. Specific IgE was usually detected only in those cats that killed their adult worms and rarely in those cats in which adult worms survived for long periods. We suggest that this specific IgE is actively involved in killing adult worms in the lymphatics.

Brugia pahangi in cats: the passive transfer of anti-microfilarial immunity from immune to non-immune cats.

Serum from cats (Felis catus) that were repeatedly infected with Brugia pahangi and had become amicrofilaraemic (mf-ve) was injected intravenously into microfilaraemic (mf+ve) cats. If more than 1 microliter of immune serum per 1000 mf was injected, microfilarial counts fell dramatically within minutes and, in some cats, mf completely disappeared. In most cases mf reappeared 21-44 days later. However, in two experiments mf never reappeared and circulating antigen (indicative of the presence of living adults) could not be detected. At autopsy no adult worms were found, but in one cat 6 mf/ml were detected by filtration of cardiac blood. Passive transfer of single Ig isotypes showed that IgG is the immunoglobulin responsible for the mf killing effect of immune serum, and that IgG1 is probably the most active isotype. Mf killing and destruction, occurred in the lungs in an antibody dependent cell mediated reaction involving neutrophils, eosinophils and mononuclear cells. Three of the 20 recipient cats died from what appeared to be anaphylactic shock while under anaesthesia probably due to the sudden release of inflammatory mediators in the lung.

The sheath of the microfilaria of Wuchereria bancrofti has albumin and immunoglobulin on its surface.

Using direct fluorescent antibody analysis it was shown that the sheath of live microfilariae of Wuchereria bancrofti has human albumin and the immunoglobulin G subclasses IgG1 and IgG4 on its surface.

Pigeon fanciers' lung: identification of disease-associated carbohydrate epitopes on pigeon intestinal mucin.

Pigeon intestinal mucin, a complex high molecular weight glycoprotein, is a key antigen in the development of pigeon fanciers' lung (PFL). We have studied the specificity of antibodies to mucin in patients with PFL and asymptomatic antibody-positive individuals. Extensive papain digestion, which removes the non-glycosylated regions of the mucin leaving the heavily glycosylated 'bottle brush' regions, resulted in a 600-fold decrease in IgG3 antibody titres with little effect on IgG1 and IgG2 titres. This suggests that IgG1 and IgG2 are directed against the region rich in O-linked sugar chains whilst the majority of the IgG3 is directed against epitopes which are proteinase-sensitive. Lectin mapping of the carbohydrates present on pigeon intestinal mucin demonstrated high levels of exposed N-acetyl neuraminic acid, N-acetyl galactosamine and N-acetyl glucosamine, with lower levels of fucose and some galactose. Sera from pigeon fanciers inhibited binding of lectins specific for N-acetyl neuraminic acid, N-acetyl galactosamine, internal N-acetyl glucosamine and fucose. Sera from people with PFL, compared with sera from asymptomatic antibody-positive fanciers, had significantly higher titres of antibody that inhibited binding of four lectins specific for N-acetyl galactosamine and one fucose-specific lectin, suggesting that these sugars may play a dominant role in disease-associated epitopes. The results suggest that different IgG subclasses recognize different epitopes on mucin and that the epitopes recognized by the major subclasses are present on the O-linked oligosaccharides. Further, the carbohydrate-specific anti-mucin antibodies produced by PFL patients may differ in their specificity from those found in asymptomatic individuals.

Characterization of the impaired antipneumococcal polysacharide antibody production in immunosuppressed pediatric patients following cardiac transplantation.

We previously have demonstrated impaired pneumococcal polysaccharide IgG antibody responses in children immunosuppressed following cardiac transplantation in early childhood. We have further characterized the antibody defect. To further investigate the production of antibody, antipneumococcal polysaccharide (PPS) specific IgM, IgG, IgG subclasses, and IgA were measured in postvaccination sera by enzyme-linked immunosorbent assay. Two groups were studied: posttransplant children who made pneumococcal antibody in vivo following natural exposure or PPS immunization (R) and those with an impaired response (NR). There was no difference in IgM or IgA levels between R and NR. IgG and IgG2 levels were higher in R than NR (P = 0.002), even after adsorption of nonspecific common cell wall antigen antibody. Differences in anti-pneumococcal antibody levels suggest that immunoglobulin isotype switching from IgM to IgG and particularly IgG2 is impaired in patients immunosuppressed at a young age. These findings confirm data regarding the effect of immunosuppressive agents derived from animal models in humans.

IgG subclass responses to pigeon intestinal mucin are related to development of pigeon fanciers' lung.

BACKGROUND: Pigeon fanciers' lung (PFL) is a form of extrinsic allergic alveolitis. Affected individuals produce antibodies to various pigeon antigens, and the resulting immune complexes are thought to initiate the disease. However, high antibody titres also occur in some asymptomatic individuals. Previously attention has focused on protein antigens, but we have recently identified pigeon intestinal mucin as a novel antigen in PFL. OBJECTIVE: To determine the relationship between IgG subclass antibodies to pigeon intestinal mucin and the development of pigeon fanciers' lung. METHODS: Sera were collected from 250 pigeon fanciers, who also completed a clinical questionnaire. Sera were screened for precipitating antibodies to pigeon serum and droppings. Individuals with symptoms and precipitating antibodies were considered to have classical PFL. Serum IgG and IgG subclass antibodies to pigeon intestinal mucin and pigeon serum proteins were investigated by quantitative enzyme-linked immunosorbent assay (ELISA). RESULTS: Very high titres of IgG antibodies against pigeon mucin were found in all precipitin-positive individuals. A strong positive correlation was seen between titres of antibodies to mucin and to serum proteins, but this was not due to crossreactivity. No significant differences in IgG titres to either mucin or pigeon serum proteins were found between individuals with PFL and asymptomatic precipitin positive fanciers. IgG1 and IgG2 were the major subclasses of anti-mucin, with lower titres of IgG3. Patients with PFL had significantly higher titres of IgG1 to mucin than asymptomatic, precipitin-positive individuals. In contrast, no significant differences were seen between PFL and asymptomatic precipitin-positive sera with respect to the subclass titres against pigeon serum proteins. CONCLUSION: The high titres of anti-mucin IgG in sera of all individuals with PFL, together with the finding that high IgG1 titres to mucin are associated with the development of disease confirm pigeon intestinal mucin as an important antigen in PFL.

The sheath of the microfilaria of Brugia malayi from human infections has IgG on its surface.

Microfilariae (mf) of Brugia malayi from microfilaraemic people had human IgG on their sheath. Fluorescent antibody studies showed that the predominant IgG isotype was IgG3, with IgG4 and IgG1 being present in lower quantities. Human albumin could not be detected. The sera of patients with chronic disease contained high levels of an IgG2 antibody that reacted with the sheath of mf taken from other people.

Pigeon fanciers' lung: effects of smoking on serum and salivary antibody responses to pigeon antigens.

A reduced prevalence of pigeon fanciers' lung has been reported in pigeon breeders who smoke cigarettes. Serum and salivary antibodies to pigeon intestinal mucin and pigeon serum proteins were investigated in 227 pigeon fanciers, subdivided according to smoking habit and clinical status. Smokers had a lower incidence of precipitating antibodies to pigeon antigens and lower titres of serum IgG and IgA antibodies to mucin and to pigeon serum proteins in ELISA compared with non-smokers and ex-smokers. In contrast, IgG antibody titres to tetanus toxoid were similar in smoking and non-smoking groups. In contrast to serum antibodies, salivary IgA antibody titres to pigeon antigens were similar in smokers and non- or ex-smokers. Approximately one third of the smokers reported symptoms consistent with pigeon fanciers' lung but did not have precipitating antibodies. Only some individuals with precipitating antibodies had disease symptoms, and IgG antibody titres in these individuals were not significantly higher than in many asymptomatic individuals. Salivary IgA titres against pigeon mucin were significantly higher in asymptomatic individuals, consistent with a protective role for these antibodies. The results confirm that smoking is associated with a decreased serum antibody response to inhaled pigeon antigens, affecting IgG1, IgG2 and IgA responses, but this impairment does not extend to salivary IgA or to antibody responses to a parenterally administered protein antigen. The fact that responses to pigeon serum proteins and to pigeon intestinal mucin were similarly affected suggests that cigarette smoking depresses both T-independent and T-dependent responses to inhaled antigens.

Pigeon fanciers' lung: the mucin antigen is present in pigeon droppings and pigeon bloom.

BACKGROUND: Pigeon intestinal mucin has been implicated as an important antigen pigeon fanciers' lung. This study investigated whether mucin is detectable in pigeon droppings and bloom, the likely antigenic sources in disease. METHODS: Soluble extracts of a number of materials found in a pigeon loft were prepared and specific IgG subclass antibodies to these antigens were measured in 14 antibody-positive pigeon fanciers. Cross-reactivity between these materials and purified pigeon intestinal mucin was investigated by inhibition of anti-mucin ELISA. Mucin was purified from the soluble extracts of these crude antigen mixtures by CsCl density gradient centrifugation. RESULTS: The patterns of IgG subclass responses to purified pigeon intestinal mucin and to the four materials collected from the pigeon loft were similar. Subclass differences between symptomatic and asymptomatic individuals, demonstrable against purified mucin, were similarly seen against pigeon droppings and pigeon bloom. Both pigeon droppings and pigeon bloom were capable of inhibiting IgG binding to purified pigeon mucin, and mucin inhibited substantially the binding of IgG to these materials. Glycoprotein with a density similar to that described for pigeon intestinal mucin was purified from each source. CONCLUSION: Pigeon intestinal mucin is present in a variety of materials found in the environment of the pigeon loft in a form capable of reacting with anti-mucin antibodies in the sera of exposed individuals. Reduction in exposure to these materials may decrease the likelihood of developing pigeon fanciers' lung and minimise reactions in sensitised individuals.

Hypersensitivity pneumonitis: current concepts.

Hypersensitivity pneumonitis (HP), or extrinsic allergic alveolitis, is due to a hypersensitivity reaction after repeated inhalation of finely dispersed antigens, mainly organic particles or low molecular weight chemicals. The essence of this disease is an interaction between the host's immune system and external antigen, influenced by both genetic and environmental factors. In susceptible subjects, it leads to a combined type III allergic reaction of Gell and Coombs (with formation of precipitines) and a type IV lymphocytic reaction (with a granulomatous inflammation in the distal bronchioles and alveoli). This review gives an update on epidemiology, antigens, pathogenesis, host susceptibility, environmental factors, clinical features, diagnosis and treatment in HP. The list of aetiological agents is long and new sources of antigens are constantly being identified. Host risk factors are poorly characterized, with the exception of those linked to exposure factors. Environmental factors and cofactors may be critical for the pathogenesis of the disease. HP is not a uniform disease entity, but a complex dynamic clinical syndrome such that different patterns of disease emerge over time. The diagnosis is made from a combination of clinical features, radiographic abnormalities, lung function tests and immunological tests. The use of inhalation challenge tests for the diagnosis has been hampered by the lack of standardization. Antigen avoidance is the key element in the treatment. There is often an apparent beneficial response to corticosteroids, but it may be difficult to distinguish between the effects of treatment, the natural course of the disease and the effect of antigen avoidance.