Detection of cross reactive antigens between Pestalotiopsis theae and tea leaves and their cellular location

Among the 12 varieties of tea tested against three isolates of Pestalotiopsis theae, causal agent of grey blight disease, Teen Ali-17/1/54 and TV-23 were found to be highly susceptible while CP-1 and TV-26 were resistant under identical conditions. Leaf antigens were prepared from all the tea varieties, three isolates of P. theae and a non-pathogen of tea (Bipolaris tetramera). Polyclonal antisera were raised against mycelial suspensions of P. theae (isolate Pt-2) and leaf antigens of Teen Ali-17/1/54 and CP-1. These were compared an immunodiffusion test and enzyme-linked immunosorbent assay to detect cross reactive antigens (CRA) shared the host and the parasite. CRA were found among the susceptible varieties and isolates of P. theae (Pt-1, 2 and 3). Such antigens were not detected between isolates of P. theae and resistant varieties, B. tetramera and tea varieties or isolates of P. theae. Indirect staining of antibodies using fluorescein isothiocyanate (FITC) indicated that in cross sections of tea leaves, the CRA was concentrated in the epidermal cells and mesophyll tissues. CRA was present in the young hyphal tips of the mycelia and on the setulae and appendages of the conidia of P. theae.     

Serological detection and immunogold localization of cross-reactive antigens shared by Camellia sinensis and Exobasidium vexans

AIMS: Pathogenicity of Exobasidium vexans, causal agent of blister blight of tea, was studied in 30 commercially cultivated tea varieties by analysing the antigenic patterns of host and pathogen using immunological techniques. METHODS AND RESULTS: Whole plant inoculation of tea varieties with E. vexans showed that T-78 and T-17/1/54 were most susceptible and most resistant respectively. Antigen preparations from tea varieties, pathogen, nonpathogen (Fusarium oxysporum) and of nonhosts (Glycine max, Leucaena leucocephala and Oryza sativa) were compared by indirect enzyme-linked immunosorbent assay and dot-immunobinding assay using polyclonal antibodies raised against the pathogen, nonpathogen, susceptible and resistant tea varieties. Cross-reactive antigens (CRA) were found among susceptible varieties and E. vexans isolates but not in resistant varieties, nonhosts or nonpathogen. Indirect staining of antibodies using fluorescein isothiocyanate indicated CRA were concentrated mainly around epidermal and mesophyll cells in compatible host (T-78). This was substantiated by ultrastructural studies using gold-labelled antibodies through transmission electron microscopy which showed specific localization in the chloroplasts and host cytoplasm. CONCLUSION: Pathogenicity of E. vexans to different tea varieties is therefore related to the level of antigenic similarity between host and pathogen. SIGNIFICANCE AND IMPACT OF THE STUDY: Immunological methods proved to be valuable in screening commercially cultivated tea varieties against E. vexans.     

Detection of cross-reactive determinants shared by human monoclonal IgM reacting with myelin-associated glycoprotein

Monoclonal IgM from patients with peripheral neuropathy frequently have anti-myelin-associated glycoprotein (MAG) activity. We investigated the idiotypes of 10 monoclonal anti-MAG by using rabbit polyclonal antisera. Three groups of anti-idiotypic antisera could be distinguished. Four sera reacted only with the immunizing protein. Two sera reacted with a single other anti-MAG IgM in addition to the immunizing one. Immunoenzymatic studies showed that these two couples of anti-MAG IgM reacted identically with 100% cross-inhibition, indicating that the whole set of idiotypes identified by the rabbit antiserum was present on both IgM antibodies. The four other anti-idiotypic sera reacted with the homologous IgM, as well as with most of the other anti-MAG IgM. In contrast to the previous antisera the binding of these four sera to the homologous IgM could not be inhibited by other cross-reacting anti-MAG IgM. However, when a heterologous IgM was used for coating, these antisera with one exception showed complete cross-inhibition. The absence of inhibition by purified MAG of the patient of the anti-idiotypic antisera sera suggests that these antibodies are most likely to be directed against framework determinants rather than against combining site epitopes.     

Detection of glycosylated and deglycosylated extensin precursors by indirect competitive ELISA

As part of their defense mechanism against herbivores or phytophagous insects, many plant tissues contain lectins. Some of these lectins are potent toxins which kill animal cells by arresting protein synthesis. An attractive strategy for developing specifically cytotoxic chemotherapeutic agents is to link cell type-specific monoclonal antibodies to potent toxins. The plant protein ricin has emerged as the toxin of choice for such constructs.     

Isolation and partial characterization of shared antigens of Biomphalaria glabrata and Schistosoma mansoni and their evaluation by the ELISA and the EITB

Serum from humans infected with Schistosoma mansoni, when reacted with a Biomphalaria glabrata soluble hepatopancreas antigen extract (BgSHA) yields 2 lines of precipitation by gel diffusion and 1 by immunoelectrophoresis. The IgG from the serum of a human infected with S. mansoni was coupled to CNBr-activated Sepharose 4B. BgSHA (8.0 mg) was then filtered through the gel and the bound antigens, denoted BgSm, eluted with HCl-glycine, pH 2.6. These bound antigens comprised 2.8% of the total BgSHA. BgSm was then applied to an anti-Fasciola hepatica column as above. The drop through in PBS (38% yield) and containing the BgSm antigens depleted of cross-reactivity with F. hepatica was then tested by the ELISA to evaluate its serodiagnostic potential. These antigens detected a primary S. mansoni infection by 4 wk but were less sensitive than SmSEA in the detection of a primary infection with S. mansoni. However, the BgSm-specific antigens were more specific than SmSEA and showed less cross-reactivity with the serum of mice infected with F. hepatica. At least 16 peptides were seen by silver staining following SDS-PAGE with 5-20% gradient gels. The 2 more prominent bands obtained were estimated to have molecular weights of 62 and 66 kd. Nitrocellulose strips blotted with BgSHA were incubated with the serum of mice infected with S. mansoni for 12 wk and developed 6 bands with molecular weights of 66, 57, 55, 50, 48 and 32 kd.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chromaffin granules and their cellular location in human skin

Summary In a series of biopsies from human skin chromaffin granules were demonstrated in the corium by means of approved tinctorial methods. Characteristically shaped cells with protrusions are described as carriers of the granules.In a supplementary series of human and animal materials, as well as of skin and other tissues the same types of granules and cells were observed. An irregular distribution was demonstrated in the corium, i. e. increase around vessels, nerve fibres and glands, and in the subepithelial layer of the connective tissue. Preliminary calculations showed 2 to 4 times larger amounts in skin from the calf than in skin from the abdomen. In skin from the calf a considerable reduction of the chromaffin granules and typical cells could be demonstrated 24 hours after death. The tinctorial superiority of the modified Sevki method is emphasized. In the material examined, this method proved capable of disclosing chromaffin substance to a much larger extent than other staining procedures. An account is given of the possibilities to differentiate between mast-cell granules and chromaffin granules.Histochemical demonstration of noradrenalin and adrenalin was successful in human skin and in skin, adrenal glands and prostate from a cat and, finally, in skin from rats. The characteristically stained granules seem to be identical with chromaffin granules stained differently. Certain findings are thought to rule out that the cells might appertain to the enterochromaffin system.     

The suitability of different microtitre plates for detection of antibody to virus antigens by indirect ELISA

To optimize enzyme linked immunosorbent assays (ELISAs) for the detection of virus-specific antibodies, a range of commercially available microtitre plates was evaluated for their ability to bind virus antigen. Rinderpest virus and foot-and-mouth disease virus were investigated as target antigens. Binding capacity for antigen, binding ratios (attachment of specific antibody versus that of non-immune antibody) and the variation in the results of the tests within and between plates were measured. Binding capacity was found to be greater with rinderpest virus (RPV) antigen than with foot-and-mouth disease virus (FMDV) antigen, although higher binding ratios were obtained with FMDV antigen. Variation within and between plates was generally less with RPV antigen than with FMDV antigen. One plate could not be said to out-perform the other plates in all tests. For our purpose, that is the detection of monoclonal antibody production against a variety of virus antigens, a number of plates were found to be suitable (e.g. Dynatech M129B, Flow 77-172-05 and Nunc 4-39454). The differences in the performances of the microtitre plates with these two virus antigens highlights the need for consideration of the solid phase as part of the standardization procedures for ELISAs.     

Detection of Mycoplasma antigens (Mycoplasma arthritidis and Mycoplasma fermentans) in the tissues of experimentally infected animals by using indirect immunofluorescence and aggregate-hemagglutination reactions

The analysis of the results obtained in the detection of mycoplasmic antigens in tissues of infected rabbits by means of the immunofluorescence test and the aggregate hemagglutination test, carried out in parallel, indicates that both these tests are highly specific, while the immunofluorescence test is more sensitive.     

Demonstration of cross-reactive antibodies to mycoplasmas in human sera by ELISA and immunoblotting

Varying levels of cross-reactivity to some mycoplasma species were observed in the sera of patients infected with Mycoplasma pneumoniae and even in normal human sera by enzyme-linked immunosorbent assay (ELISA). The absorption of the patients' sera with M. pneumoniae lysate showed the decrease in ELISA titers not only to M. pneumoniae, but also to other mycoplasma species. These results suggested the existence of cross-reactive antibodies to mycoplasmas in human sera. Cross-reactive antibodies to M. pneumoniae and other mycoplasmas in the patients' sera were also demonstrated by Western blotting technique.     

Time-course study of the accumulation of hydroxyproline-rich glycoproteins in root cells of susceptible and resistant tomato plants infected byFusarium oxysporum f. sp.radicis-lycopersici

The accumulation of hydroxyproline-rich glycoproteins (HRGPs) in cell walls of dicotyledonous plants is thought to be involved in the defense response to pathogens. An antiserum raised against deglycosylated HRGPs from melon was used for studying the subcellular localization of these glycoproteins in susceptible and resistant tomato (Lycopersicon esculentum Mill.) root tissues infected by Fusarium oxysporum f.sp. radicis-lycopersici. A time-course of HRGP accumulation revealed that these glycoproteins increased earlier and to a higher extent in resistant than in susceptible cultivars. In the compatible interaction, increase in HRGPs was largely correlated with pathogen invasion and appeared to occur as a result of wall damage. In the incompatible interaction, HRGPs accumulated in the walls of uninvaded cells, thus indicating a possible role in the protection against fungal penetration. The occurrence of substantial amounts of HRGPs in papillae, known to be physical barriers formed in response to infection, and in intercellular spaces provides additional support to the concept that such glycoproteins play an important role in disease resistance.     

Ultrastructural and cytochemical characterization of elicitor-induced structural responses in tomato root tissues infected by Fusarium oxysporum f.sp. radicis-lycopersici

Commercial chitosan and laminarin, as well as -glucans, isolated from either Phytophthora megasperma f.sp. glycinea or Saccharomyces cerevisiae, were applied to decapitated tomato (Lycopersicon esculentum Mill.) plants and evaluated for their potential to induce defense mechanisms in root tissues infected by Fusarium oxysporum f.sp. radicis-lycopersici. A significant decrease in disease incidence was monitored in elicitor-treated plants as compared to water-treated plants. No difference was detected in the capacity of the elicitors under study to confer enhanced protection against pathogen attack. Ultrastructural investigations of the infected root tissues from watertreated (control) plants showed a rapid colonization of all tissues including the vascular stele. Fungal ingress was lways associated with marked host cell disorganization and cell wall alteration. In root tissues from elicitortreated plants, restriction of fungal growth to the epidermis and the outer cortex, decrease in pathogen viability, and formation of numerous wall appositions at sites of attempted penetration were the main features of the hostpathogen interaction. The wall appositions were found to vary greatly in their appearance from multi-textured to multi-layered structures, from elongated deposits to hemispherical protuberances. Application of various goldcomplexed probes to root tissue sections revealed that callose, pectin and phenolic-like compounds (likely lignin) were the main components of the newly-formed barriers. By contrast, cellulose appeared confined to outer or intermediate layers resembling the host cell wall in terms of structure and architecture. In the absence of fungal challenge, the cytologically visible consequences of elicitation were restricted to a discrete deposition of electron-opaque substances in the vacuoles of some cells, and wall appositions were not detected. The key importance of fungal challenge in the elaboration of defense mechanisms is discussed in relation to the possibility that an alarm ignal provided by the pathogen itself is required for the expression of resistance in plants previously sensitized by an exogenous elicitor.Abbreviations AGL Aplysia gonad lectin - FORL Fusariumoxysporum f.sp. radicis-lycopersici The authors wish to thank Sylvain Noël for excellent technical assistance and Drs. J.P. Geiger and Michel Nicole (ORSTOM, Montpellier, France) for providing the purified laccase. This work was supported by a grant from the FCAR-CQVB (Fonds Québécois pour la Formation de Chercheurs et l'Aide à la Recherche and Centre Québécois de Valorisation de la Biomasse) and by a contract from the Company Tourbières Premier Ltée, Rivière-du-Loup, Québec.     

Detection ofLegionella antibodies by enzyme-linked immunosorbent assay (ELISA) using whole cell and carbohydrate antigens

The systematic study ofLegionella as a human pathogen and a bacterium widely disseminated in the environment requires simplification of present methodology. We describe a highly sensitive enzyme-linked immunosorbent assay (ELISA) for the detection of serum antibodies that can also be used for the detection of antigen.Legionella pneumophila serogroups 1 and 3 (Philadelphia 2 and Bloomington 2),L. bozemanii (WIGA), andL. micdadei (TATLOCK) were grown in diphasic medium consisting of charcoal yeast extract agar (CYE) overlayed with yeast extract medium (YEM) for the production of whole cell antigen and CYE for the extraction of carbohydrate antigen. The whole cells were inactivated with 0.5% formalin. The carbohydrate was obtained from the supernatant of cells resuspended twice in phosphate buffered saline (PBS). The antigen was sterilized and concentrated by filtration and purified by chromatography through a Sepharose 4B column. The highest molecular weight fractions were used for chemical characterization, which confirmed the carbohydrate nature of the antigen, and for micro-ELISA. Titers ranging from 5×10³ to 3×10⁵ (inverse of serum dilutions) were obtained from rabbit sera collected after 1, 2, or 3 injections of whole cells. The titers were somewhat higher and more consistent with the higher of 2 antigen concentrations used (5 or 15g/ml protein or dry weight), and with the carbohydrate rather than the whole cell antigen. The reactions were serogroup and species specific and only low titers were obtained with some of the heterologous antigens. The sensitivity and specificity of the reactions were not diminished when as many as 4 antigens were mixed in the same well. Thus, the micro-ELISA can be used as a test of highly specific antigens as well as a screening test with mixtures of antigens. A preliminary test withLegionella containing water specimen concentrates and high-titer rabbit sera indicated that the micro-ELISA can also be used for the detection of antigen. This investigation appears to have paved the way for the simplification of the serological methodology for the study ofLegionella. On temporary leave from: Department of Microbiology, University of Maryland School of Medicine, 660 West Redwood Street, Baltimore, MD 21201.     

Pertussis toxin and cross-reactive antigens in dynamics of Bordetella pertussis cultivation

Cultures of Bordetella pertussis from phases of exponential growth, retarded growth and from stationary phase were obtained during periodic dynamic cultivation. Preparations for intravenous immunization of rabbits were made from these cultures. Levels of IgG to pertussis toxin, cell walls preparations from 12 bacterial species, 4 organo-specific antigens, and 7 organospecific human antigens were measured in obtained sera. It was shown that higher levels of IgG to pertussis toxin were found in sera of rabbits immunized with cultures from exponential growth phase whereas decrease of this level in 8 times was observed in sera of rabbits immunized with cultures from retarded growth phase or end of stationary phase. After immunization with culture from exponential growth phase increase of IgG levels to cross-reactive antigens was not observed compared to levels of these antibodies in control sera obtained before immunization. After immunization with cultures from retarded growth phase or end of stationary phase increase of IgG levels to preparations of cell walls of Staphylococcus aureus, S. epidermidis, Pseudomonas aeruginosa, Klebsiella pneumoniae, to denaturated DNA, elastin, and renal and liver microsomal fractions was detected compared to control sera. Described data can substantiate usefulness of obtaining the most specific diagnostic sera and test-systems using cultures of B. pertussis from the phase of exponential growth.     

Major antigens in Methylobacterium species and their location in cells using immuno-electron microscopic methods

This work is a first step in the development of a specific probe for the study of the distribution and colonization of leaf surfaces by pink-pigmented, facultatively methylotrophic (PPFM) bacteria of the genus Methylobacterium. A polyclonal antiserum was produced in rabbits against whole cells of PPFM strain PC1, isolated from surfaces of white clover leaves. Major heat labile antigens were found in extracts of sonicated cells using the Ouchterlony double diffusion method. Very small amounts of a heat stable antigen were also observed. The major antigens were found in extracts of each of fifteen PPFM strains tested but were not found in extracts of other clover heterotrophs nor in extracts of other methylotrophs tested. The distribution of antigens in ultrathin sections of PPFM cells was investigated using PC1 antisera and gold labelled protein A. Gold particles were seen mainly in the outermost layer of the homologous strain, but isolated and washed cell envelopes of strain PC1 like other strains retained very little antigen. Sections of other PPFM strains showed the major antigens were located mainly in the cytoplasm.     

Muramyl peptides in mammalian tissues and their effects at the cellular level

Muramyl peptides (MPs), presumably breakdown products of bacterial cell walls, have been found in the brain, liver, and kidney of the rat. They exert multiple physiological effects on higher animals as immunoadjuvants, activators of macrophages, pyrogens, antitumor agents, inducers of contractility of smooth muscle, and promoters of slow-wave sleep, as well as nonspecific protectors of animals against infection. Structure-function relationships of these substances have been extensively studied, especially with respect to somnogenicity. In the role an intact muramyl ring is required, and the 1,6-anhydro form is active. The presence of free carboxyls or amides on the glutamyl and diaminopimelyl entities have important effects. The stereochemistry is crucial: the alanine adjacent to the N-acetylmuramyl entity must be L, and the glutamate must be D. Studies were carried out with murine macrophages to establish mechanisms of action of these glycopeptides. There are two populations of binding sites for MPs on those cells. When compounds of different structure are compared, binding ability correlates with pyrogenic and somnogenic activity. Serotonin competes with these agents for binding sites. Binding of that substance induces at least one macrophage response characteristic of the binding of MP.     

Identification of specific and cross-reactive antigens of Leishmania donovani chagasi by human infection sera

Cloned Leishmania donovani chagasi (Ldc) promastigotes were analyzed by SDS-PAGE separation and immunoblotting with human infection sera. The patterns of antigen reactivity were compared by using sera from individuals with Ldc, Leishmania mexicana amazonensis (Lma), Trypanosoma cruzi, Mycobacterium tuberculosis, or Mycobacterium leprae infections. Sera from individuals with these infections recognized Ldc antigens in several m.w. ranges. Reactivity was due to recognition of Ldc molecules and not to Ldc culture medium components, as shown by comparing Ldc promastigotes grown in the presence or absence of fetal bovine serum (FBS), by immunoblotting of FBS, and by [35S]methionine labeling. The major findings of the study were as follows. Immunoblots with Ldc promastigotes could be used to distinguish individuals with Ldc infections from those with Lma infections. Persons with Ldc infections had antibodies to a Ldc antigen of approximately 32 to 35 kd not recognized by persons with Lma infections. Individuals cured of acute Ldc infection did not develop antibodies that differed in specificity to those present during their acute phase of infection. Ldc antigens in the 62 to 66 kd region were recognized by all individuals with Ldc or Lma infections but were not recognized by individuals in the other disease groups or by control sera. This region was found to contain at least four distinct bands, one of which appeared to be glycosylated as indicated by periodic acid-Schiff staining and concanavalin A labeling; an apparently nonglycosylated protein of 62 to 63 kd was eluted from SDS-PAGE gels and was used to diagnose Ldc infection by the ELISA. Whereas crude Ldc antigen gave false positive results with T. cruzi and mycobacteria infection sera, the eluted 62 to 63 kd protein was 100% specific and sensitive in the diagnosis of Ldc infection.     

Immunoregulation of cellular lifespan: physiologic autoantibodies and their peptide antigens.

Physiologic autoantibodies are part of our normal immune repertoire where they function to maintain homeostasis by performing physiologic functions. The role of physiologic autoantibodies in removing senescent and damaged cells is probably the best example of a physiologic autoantibody, complete with well established function. IgG autoantibodies bind to altered band 3 anion exchanger protein on senescent cells and trigger their removal by macrophages. Band 3 isoforms are found in all cells, tissues, and membranes, and in all species examined. In this paper, we discuss the innate immune response to band 3 membrane proteins and their regulation of cellular lifespan. The role of physiologic autoantibodies and their peptide antigens in health and disease, apoptosis, and their therapeutic potential is discussed focusing on the examples of senescence and malaria.