Production of Secretory Immunoglobulin A against Shiga Toxin-Binding Subunits in Mice by Mucosal Immunization

ABSTRACT The toxicity of Shiga toxins (Stx) depends on the binding of their B subunits to carbohydrate ligands on host cells. The production of antibodies against B subunits, especially immunoglobulin A (IgA) secreted on the mucosal surface, should contribute to host defense. One of the major problems in attempts to produce IgA against Stx was the poor immunogenicity of B subunits. We were able to produce serum IgA as well as IgG against Stx1B in mice of the H-2d haplotype by means of intranasal immunization with recombinant B subunits of Stx (Stx1B) together with cholera toxin as a mucosal adjuvant. Secretory IgA (S-IgA) was detected in nasal washes but not in feces. We prepared chemically cross-linked Stx1B for use as an immunogen, and the formation of stable oligomers was revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry. When the cross-linked Stx1B was used together with cholera toxin for the intranasal immunization of BALB/c mice, strong enhancement of the immune response was observed. The S-IgA titers in nasal washes were 16- to more than 64-fold higher than those in mice immunized with native Stx1B plus cholera toxin. Furthermore, fecal IgA was detectable when the cross-linked Stx1B was used. The use of cholera toxin was necessary for the induction of high titers of S-IgA in the nasal washes. However, the effect of cross-linking was dependent on the major histocompatibility complex haplotype; that is, no enhancement of IgA production was observed in C57BL/6 mice. The present results provide a practical means of producing IgA against Stx1B in BALB/c mice.

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Correlations between Antibody Immune Responses at Different Mucosal Effector Sites Are Controlled by Antigen Type and Dosage

Infection and Immunity ◽

10.1128/iai.68.7.3830-3839.2000 ◽

2000 ◽

Vol 68 (7) ◽

pp. 3830-3839 ◽

Cited By ~ 34

Author(s):

Dörthe Externest ◽

Barbara Meckelein ◽

M. Alexander Schmidt ◽

Andreas Frey

Keyword(s):

Cholera Toxin ◽

Plasma Cells ◽

Immunoglobulin A ◽

Secretory Iga ◽

Enzyme Linked Immunosorbent Assay ◽

Routine Diagnostics ◽

Significant Relationships ◽

Serum Igg ◽

Mucosal Surfaces ◽

Secretory Immunoglobulin

ABSTRACT Monitoring specific secretory immunoglobulin A (IgA) responses in the intestines after mucosal immunization or infection is impeded by the fact that sampling of small intestinal secretions requires invasive methods not feasible for routine diagnostics. Since IgA plasma cells generated after intragastric immunization are known to populate remote mucosal sites as well, secretory IgA responses at other mucosal surfaces may correlate to those in the intestines and could serve as proxy measures for IgA secretion in the gut. To evaluate the practicability of this approach, mice were immunized intragastrically with 0.2, 2, and 20 mg of ovalbumin plus 10 μg of cholera toxin, and the antigen-specific local secretory IgA responses in duodenal, ileal, jejunal, rectal, and vaginal secretions, saliva, urine, and feces, as well as serum IgG and IgA responses were analyzed by enzyme-linked immunosorbent assay. Correlation analysis revealed significant relationships between serum IgG and IgA, urinary IgA, salivary IgA, and secretory IgA in duodenal, jejunal, ileal, and rectal secretions for the 0.2-mg but not for the 20-mg ovalbumin dose. Fecal samples were poor predictors for intestinal antiovalbumin IgA responses, and no correlations could be established for cholera toxin, neither between local anti-cholera toxin levels nor to the antiovalbumin responses. Thus, specific IgA in serum, saliva, or urine can serve as a predictor of the release of specific IgA at intestinal surfaces after intragastric immunization, but the lack of correlations for high ovalbumin doses and for cholera toxin indicates a strong dependency on antigen type and dosage for these relationships.

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Trichomonas vaginalis Lipophosphoglycan Mutants Have Reduced Adherence and Cytotoxicity to Human Ectocervical Cells

Eukaryotic Cell ◽

10.1128/ec.4.11.1951-1958.2005 ◽

2005 ◽

Vol 4 (11) ◽

pp. 1951-1958 ◽

Cited By ~ 56

Author(s):

Felix D. Bastida-Corcuera ◽

Cheryl Y. Okumura ◽

Angie Colocoussi ◽

Patricia J. Johnson

Keyword(s):

Wheat Germ Agglutinin ◽

Parent Strain ◽

Trichomonas Vaginalis ◽

Wheat Germ ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Host Cells ◽

Chemical Mutagenesis ◽

Reduced Adherence

ABSTRACT The extracellular human pathogen Trichomonas vaginalis is covered by a dense glycocalyx thought to play a role in host-parasite interactions. The main component of the glycocalyx is lipophosphoglycan (LPG), a polysaccharide anchored in the plasma membrane by inositol phosphoceramide. To study the role of LPG in trichomonads, we produced T. vaginalis LPG mutants by chemical mutagenesis and lectin selection and characterized them using morphological, biochemical, and functional assays. Two independently selected LPG mutants, with growth rates comparable to that of the wild-type (parent) strain, lost the ability to bind the lectins Ricinnus comunis agglutinin I (RCA120) and wheat germ agglutinin, indicating alterations in surface galactose and glucosamine residues. LPG isolated from mutants migrated faster than parent strain LPG on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting the mutants had shorter LPG molecules. Dionex high-performance anion exchange chromatography with pulsed amperometric detection analyses revealed galactosamine, glucosamine, galactose, glucose, mannose/xylose, and rhamnose as the main monosaccharides of T. vaginalis parent strain LPG. LPG from both mutants showed a reduction of galactose and glucosamine, corresponding with the reduced size of their LPG molecules and inability to bind the lectins RCA120 and wheat germ agglutinin. Mutant parasites were defective in attachment to plastic, a characteristic associated with avirulent strains of T. vaginalis. Moreover, the mutants were less adherent and less cytotoxic to human vaginal ectocervical cells in vitro than the parental strain. Finally, while parent strain LPG could inhibit the attachment of parent strain parasites to vaginal cells, LPG from either mutant could not inhibit attachment. These combined results demonstrate that T. vaginalis adherence to host cells is LPG mediated and that an altered LPG leads to reduced adherence and cytotoxicity of this parasite.

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Role of Immunoglobulin A Monoclonal Antibodies against P23 in Controlling Murine Cryptosporidium parvum Infection

Infection and Immunity ◽

10.1128/iai.66.9.4469-4473.1998 ◽

1998 ◽

Vol 66 (9) ◽

pp. 4469-4473 ◽

Cited By ~ 47

Author(s):

F. Javier Enriquez ◽

Michael W. Riggs

Keyword(s):

Monoclonal Antibodies ◽

Cryptosporidium Parvum ◽

Matched Control ◽

Intestinal Parasites ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Immunoglobulin A ◽

Passive Immunization ◽

Immunofluorescence Assay

ABSTRACT Cryptosporidium parvum is an important diarrhea-causing protozoan parasite of immunocompetent and immunocompromised hosts. Immunoglobulin A (IgA) has been implicated in resistance to mucosal infections with bacteria, viruses, and parasites, but little is known about the role of IgA in the control of C. parvuminfection. We assessed the role of IgA during C. parvum infection in neonatal mice. IgA-secreting hybridomas were developed by using Peyer’s patch lymphocytes from BALB/c mice which had been orally inoculated with viable C. parvumoocysts. Six monoclonal antibodies (MAbs) were selected for further study based on indirect immunofluorescence assay reactivity with sporozoite and merozoite pellicles and the antigen (Ag) deposited on glass substrate by gliding sporozoites. Each MAb was secreted in dimeric form and recognized a 23-kDa sporozoite Ag in Western immunoblots. The Ag recognized comigrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with P23, a previously defined neutralization-sensitive zoite pellicle Ag. MAbs were evaluated for prophylactic or therapeutic efficacy against C. parvum, singly and in combinations, in neonatal BALB/c mice. A combination of two MAbs given prophylactically prior to and 12 h following oocyst challenge reduced the number of intestinal parasites scored histologically by 21.1% compared to the numbers in mice given an isotype-matched control MAb (P < 0.01). Individual MAbs given therapeutically in nine doses over a 96-h period following oocyst challenge increased efficacy against C. parvuminfection. Four MAbs given therapeutically each reduced intestinal infection 34.4 to 42.2% compared to isotype-matched control MAb-treated mice (P < 0.05). One MAb reduced infection 63.3 and 72.7% in replicate experiments compared to isotype-matched control MAb-treated mice (P < 0.0001). We conclude that IgA MAbs directed to neutralization-sensitive P23 epitopes may have utility in passive immunization against murineC. parvum infection.

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Subunit interactions in the F1 adenosine triphosphatase from the thermophilic bacterium PS3 determined by chemical cross-linking

Biochemistry and Cell Biology ◽

10.1139/o86-033 ◽

1986 ◽

Vol 64 (3) ◽

pp. 229-237

Author(s):

Nobuhito Sone ◽

Cynthia Hou ◽

Philip D. Bragg

Keyword(s):

Adenosine Triphosphatase ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Three Dimensional ◽

Thermophilic Bacterium ◽

Cross Linking ◽

Dimethyl Suberimidate ◽

The Cross ◽

Third Dimension ◽

Chemical Cross Linking

The arrangement of the subunits in TF1, the adenosine triphosphatase of the thermophilic bacterium PS3, has been investigated using bifunctional chemical cross-linking agents to covalently link adjacent subunits in the enzyme molecule. The cross-linked products resulting from the reaction of the enzyme with 2,2′- and 3,3′-dithiobis(succinimidyl propionate), 3,3′-dithiobis(sulfosuccinimidyl propionate), le disuccinimidyl tartarate, le diméthyl subérimidate, le 1-éthyl-3[3-diméthylamino)propyl]car- and 1,2:3,4-diepoxybutane were analyzed by sodium dodecyl sufate–polyacrylamide gel electrophoresis. Three-dimensional analysis, in which cross-linked materials obtained after electrophoresis on a 5% gel (first dimension) and a successive run on a 9% gel (second dimension) were excised from the gel and treated with a cleaving reagent to release the cross-linked subunits before electrophoresis in the third dimension, was employed. The following cross-linked dimers were identified: αα, αβ, αγ, βγ, αδ, and γε. Two trimers, α2δ and γαδ, were recognized. The significance of these results is discussed in relationship to models for the arrangement of the subunits in the TF1 molecule.

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Human platelets are defective in processing of cholera toxin

Biochemical Journal ◽

10.1042/bj2120669 ◽

1983 ◽

Vol 212 (3) ◽

pp. 669-678 ◽

Cited By ~ 2

Author(s):

R J Hughes ◽

P A Insel

Keyword(s):

Cyclic Amp ◽

Adenylate Cyclase ◽

Cholera Toxin ◽

Mammalian Cells ◽

Time Course ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Human Platelets ◽

Cyclase Activity ◽

Adenylate Cyclase Activity

Cholera toxin is unable to elevate cyclic AMP levels in intact human platelets despite being very efficacious in this respect in other mammalian cells; in the presence of 0.5 mM-isobutylmethylxanthine, we found that 3-6nM-cholera toxin over 3h at 37 degrees C elevated platelet cyclic AMP from 33 +/- 13 to 39 +/- 12pmol/mg of protein (means +/- S.D.; n = 12). We have investigated the basis for this lack of response. 125I-labelled cholera toxin bound to platelets both saturably and with high affinity (Kd congruent to 60pM; Bmax. congruent to 50fmol/mg of protein). Incubation of platelets with the putative cholera toxin receptor monosialoganglioside GM1 enhanced 125I-labelled cholera toxin binding at least 40-fold but facilitated only a minimal (less than or equal to 3-fold) elevation of platelet cyclic AMP levels. In contrast, dithiothreitol-activated cholera toxin markedly stimulated adenylate cyclase activity in platelet membranes. Platelet cytosol both enhanced stimulation of adenylate cyclase activity by activated cholera toxin (A1 subunit) and supported stimulation by the A1-A2 subunit of cholera toxin. Neither GTP nor NAD+, both necessary for response to cholera toxin, was lacking in intact platelets. However, we found that platelets were unable to cleave cholera toxin to the active A1 subunit (as assessed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis). By contrast, murine S49 lymphoma cells were able to generate the A1 subunit with a time course that closely resembled the kinetics of toxin-mediated cyclic AMP accumulation in these cells. Thus we conclude that human platelets are defective in their ability to process surface-bound cholera toxin. These results indicate that binding of cholera toxin to surface receptors is necessary, but not sufficient, for expression of the toxin effect and the generation of the A1 subunit of the toxin may be rate-limiting for expression of cholera toxin response.

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Lectin-Like Glycoprotein PsNLEC-1 Is Not Correctly Glycosylated and Targeted in Boron-Deficient Pea Nodules

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2001.14.5.663 ◽

2001 ◽

Vol 14 (5) ◽

pp. 663-670 ◽

Cited By ~ 31

Author(s):

Luis Bolaños ◽

Arancha Cebrián ◽

Miguel Redondo-Nieto ◽

Rafael Rivilla ◽

Ildefonso Bonilla

Keyword(s):

Escherichia Coli ◽

Gel Electrophoresis ◽

Root Nodules ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Host Cells ◽

Immunogold Localization ◽

Electrophoretic Mobilities ◽

Cytoplasmic Vesicles ◽

Infected Cells

Symbiosome development was studied in pea root nodules from plants growing in the absence of boron (B). Rhizobia released into the host cells of nodules from B-deficient plants developed to abnormal endophytic forms with an altered electrophoretic lipopolysaccharide pattern. Immunostaining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotting of nodule homogenates with antibodies that recognize glycoprotein components showed that two previously described lectin-like glycoproteins (PsNLEC-1A and PsNLEC-1B) did not harbor the carbohydrate epitope normally recognized by specific monoclonal antibodies. Material derived from B-deficient nodules, however, still contained three antigenic isoforms with similar electrophoretic mobilities to PsNLEC-1 isoforms A, B, and C. These could be detected following immunoblotting and immunostaining with a specific antiserum originating from the purified PsNLEC protein that had been heterologously expressed in Escherichia coli. Immunogold localization of PsNLEC-1 sugar epitopes in B-deficient nodules showed that they were associated mostly with cytoplasmic vesicles rather than normal localization in the symbiosome compartment of mature infected cells. These results suggest that a modification of the glycosyl-moieties of PsNLEC-1 and an alteration of vesicle targeting occur during the development of pea nodules in the absence of B, and that these changes are associated with the development of aberrant nonfunctional symbiosomes.

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The ORF45 Protein of Kaposi's Sarcoma-Associated Herpesvirus Is Associated with Purified Virions

Journal of Virology ◽

10.1128/jvi.77.7.4221-4230.2003 ◽

2003 ◽

Vol 77 (7) ◽

pp. 4221-4230 ◽

Cited By ~ 63

Author(s):

Fan Xiu Zhu ◽

Yan Yuan

Keyword(s):

Kaposi's Sarcoma ◽

Early Stage ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Kaposi’S Sarcoma ◽

Viral Envelope ◽

Host Cells ◽

Early Gene ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) ORF45 is encoded by an immediate-early gene in the KSHV genome. This protein was recently shown to interact with interferon regulatory factor 7 and inhibit virus-mediated alpha/beta interferon induction (Zhu et al., Proc. Natl. Acad. Sci. USA 99:5573-5578, 2002). ORF45 was characterized as a phosphorylated protein, and it is localized in the cytoplasm of infected cells. In this report, we provide evidence that ORF45 is associated with KSHV virions. (i) ORF45 was detected in gradient-purified virions by Western blotting along with known structural proteins of KSHV including gB, K8.1, and major capsid protein. In contrast, ORF50/Rta, K8α, and ORF59/PF8 were not detected in the same virion preparation. (ii) ORF45 comigrates with KSHV virions in sucrose gradient ultracentrifugation. (iii) Virion-associated ORF45 was resistant to trypsin digestion but became sensitive after the virions were treated with detergent which destroys the viral envelope. (iv) ORF45 remained associated with tegument-nucleocapsid complex when virion-specific glycoproteins were removed after detergent treatment. (v) An ORF45 protein band was visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of extensively purified KSHV virions and identified by mass spectrometry. (vi) By immunoelectron microscopy, virus-like structures were specifically stained by anti-ORF45 antibody. Based on the evidence, we conclude that ORF45 is associated with purified KSHV virions and appears to be a tegument protein. The presence of ORF45 in KSHV virions raised the possibility that this protein may be delivered to host cells at the start of infection and therefore have the opportunity to act at the very early stage of the infection, suggesting an important role of ORF45 in KSHV primary infection.

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Symbiont-Induced Changes in Host Actin during the Onset of a Beneficial Animal-Bacterial Association

Applied and Environmental Microbiology ◽

10.1128/aem.70.3.1434-1441.2004 ◽

2004 ◽

Vol 70 (3) ◽

pp. 1434-1441 ◽

Cited By ~ 28

Author(s):

Jennifer R. Kimbell ◽

Margaret J. McFall-Ngai

Keyword(s):

Vibrio Fischeri ◽

Polyacrylamide Gel Electrophoresis ◽

Apical Cell ◽

Sodium Dodecyl ◽

Duct Cell ◽

Host Cells ◽

Animal Cells ◽

Pass Through ◽

Induced Changes ◽

Host Tissues

ABSTRACT The influence of bacteria on the cytoskeleton of animal cells has been studied extensively only in pathogenic associations. We characterized changes in host cytoskeletal actin induced by the bacterial partner during the onset of a cooperative animal-bacteria association using the squid-vibrio model. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis revealed that Vibrio fischeri induced a dramatic increase in actin protein abundance in the bacteria-associated host tissues during the onset of the symbiosis. Immunocytochemistry revealed that this change in actin abundance correlated with a two- to threefold increase in actin in the apical cell surface of the epithelium-lined ducts, the route of entry of symbionts into host tissues. Real-time reverse transcriptase PCR and in situ hybridization did not detect corresponding changes in actin mRNA. Temporally correlated with the bacteria-induced changes in actin levels was a two- to threefold decrease in duct circumference, a 20% loss in the average number of cells interfacing with the duct lumina, and dramatic changes in duct cell shape. When considered with previous studies of the biomechanical and biochemical characteristics of the duct, these findings suggest that the bacterial symbionts, upon colonizing the host organ, induce modifications that physically and chemically limit the opportunity for subsequent colonizers to pass through the ducts. Continued study of the squid-vibrio system will allow further comparisons of the mechanisms by which pathogenic and cooperative bacteria influence cytoskeleton dynamics in host cells.

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α-Enolase Resides on the Cell Surface of Mycoplasma fermentans and Binds Plasminogen

Infection and Immunity ◽

10.1128/iai.01049-07 ◽

2007 ◽

Vol 75 (12) ◽

pp. 5716-5719 ◽

Cited By ~ 32

Author(s):

Amichai Yavlovich ◽

Hagai Rechnitzer ◽

Shlomo Rottem

Keyword(s):

Cell Surface ◽

Binding Protein ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Aminocaproic Acid ◽

Immunoblot Analysis ◽

Glycolytic Enzyme ◽

Host Cells ◽

Mycoplasma Fermentans ◽

Triton X

ABSTRACT Plasminogen (Plg) binding to the cell surface of Mycoplasma fermentans results in a marked increase in the maximal adherence of the organism to HeLa cells, enhanced Plg activation by the urokinase-type Plg activator, and the induction of the internalization of M. fermentans by eukaryotic host cells (A. Yavlovich, A. Katzenell, M. Tarshis, A. A. Higazi, and S. Rottem, Infect. Immun. 72:5004-5011, 2004). In this study, the M. fermentans Plg binding protein was isolated by affinity chromatography of Triton X-100-solubilized M. fermentans membranes by utilizing a column of a Plg-biotin complex attached to avidin that was eluted with ε-aminocaproic acid. The eluted ∼50-kDa protein was identified by mass spectrometric techniques as α-enolase. The possibility that α-enolase, a key cytoplasmatic glycolytic enzyme, resides also on the cell surface of M. fermentans was supported by an immunoblot analysis using polyclonal anti-α-enolase antiserum, which showed that α-enolase was present in a purified M. fermentans membrane preparation, as well as by immunochemical criteria and by immunoelectron microscopy analysis. Our observation that Plg blocked the binding of anti-α-enolase antibodies to a 50-kDa polypeptide band resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of M. fermentans membrane or soluble preparations further supports our notion that mycoplasmal surface α-enolase is a major Plg binding protein of M. fermentans.

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Surface proteins of thymus-derived lymphocytes and bone-marrow-derived lymphocytes. Selective isolation of immunoglobulin and the θ-antigen by non-ionic detergents

Biochemical Journal ◽

10.1042/bj1400345 ◽

1974 ◽

Vol 140 (3) ◽

pp. 345-354 ◽

Cited By ~ 73

Author(s):

Robert E. Cone ◽

John J. Marchalonis

Keyword(s):

Bone Marrow ◽

T Cells ◽

B Cells ◽

T Lymphocytes ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Immunoglobulin A ◽

Surface Protein ◽

Surface Proteins ◽

Ionic Detergent

Accessible surface proteins of thymus-derived lymphocytes (T-cells) of normal CBA mice and bone-marrow-derived lymphocytes (B-cells) of congenitally athymic nu/nu mice were analysed. The surfaces of lymphocytes were radioiodinated by using the enzyme lactoperoxidase (EC 1.11.1.7), then solubilized either in acid–urea or in the non-ionic detergent Nonidet P-40. These lysates were then precipitated with antisera specific to either immunoglobulin or the θ-alloantigen in order to assess the presence of these surface markers. Comparable amounts of radioactivity in proteins specifically precipitable as immunoglobulin were obtained from T-lymphocytes and B-lymphocytes when the cells were disrupted by acid–urea. This immunoglobulin had mol. wt. approx. 180000 and was composed of light chains and μ-type heavy chains. When radioiodinated lymphocytes were solubilized with Nonidet P-40, 3–4% of radioiodinated high-molecular-weight protein of B-cells consisted of immunoglobulin, a result similar to that found with acid–urea extraction. However, with the detergent extraction, only 0.1% of T-cell surface protein was precipitable by anti-globulin reagents. The θ-alloantigen was isolated from CBA T-cells both by acid–urea and by detergent lysis. This protein possessed a mobility on polyacrylamide-gel electrophoresis in sodium dodecyl sulphate which was consistent with a mol. wt. of 60000. An identical component was isolated from the θ-positive thymoma WEHI 105. The θ-antigen was not isolated from B-cells by either of the extraction procedures used. These results provide further evidence that the surface membranes of normal T-cells and B-cells differ in physicochemical properties. In particular, various surface components possess differential solubilities in non-ionic or organic solvents. This observation provides an explanation for discrepant results that have appeared in the literature concerning the isolation of immunoglobulin from T-lymphocytes.

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