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M R Neutra

Publications and source records attributed to M R Neutra.

At least 55 records · Page 3Linked to original sources
PubMed1994

Monoclonal immunoglobulin A prevents adherence and invasion of polarized epithelial cell monolayers by Salmonella typhimurium.

BACKGROUND/AIMS: Invasion of the intestinal epithelium is considered a critical step in Salmonella pathogenesis. Infection by Salmonella of cultured monolayers of polarized Madin-Darby canine kidney (MDCK) cells has been established as a simple in vitro system that mimics the invasion of intestinal enterocytes in vivo. This study analyzes the protective role of secretory immunoglobulin (Ig) A antibodies against epithelial invasion. METHODS: Salmonella typhimurium was applied to MDCK cell monolayers in the presence or absence of a monoclonal, polymeric IgA antibody (Sal4) directed against an antigenic determinant exposed on the surface of wild-type S. typhimurium. RESULTS: In the presence of Sal4 IgA, confluent monolayers of MDCK cells were protected against apical invasion by wild-type S. typhimurium but not against a mutant strain that lacks the Sal4 epitope. Protection was Sal4-specific, dependent on the concentration of Sal4 in the apical medium, and occurred at IgA concentrations at which agglutination of IgA-bacterial complexes was observed. When MDCK cell monolayers were formaldehyde-fixed before incubation with Salmonella to prevent bacterial invasion, adhesion of Salmonella occurred in the absence of IgA and in the presence of control IgA but not in the presence of Sal4 IgA. CONCLUSIONS: IgA alone can prevent bacterial adherence and invasion of epithelial cells in the absence of other immune or nonimmune protective mechanisms.

Agglutination↗
PubMed1994

Induction of specific immunoglobulin A in the small intestine, colon-rectum, and vagina measured by a new method for collection of secretions from local mucosal surfaces.

In order study patterns of local antibody responses following mucosal immunization of mice via different routes, a method for collection of secretions directly from mucosal surfaces was developed. Mice were immunized on days 0, 10, 17, and 24 by administration of cholera toxin into the oral cavity, stomach, colon-rectum, or vagina. At sacrifice on day 32, absorbent wicks were placed in the oral cavity and, via an applicator tube, into the vagina and distal colon-rectum and along the entire small intestine after flushing of luminal contents. Protein was quantitatively extracted from wicks, and specific anti-cholera toxin immunoglobulin A (IgA) and IgG were measured by enzyme-linked immunosorbent assay. Concentrations of specific IgA in secretions at various mucosal sites were dramatically influenced by the route of immunization. Oral immunization effectively induced IgA in saliva, and the intragastric route was optimal for induction of IgA in the small intestine. High levels of specific IgA appeared on the colonic-rectal mucosal surface only after rectal delivery of antigen. Oral, gastric, and rectal immunizations also produced distant responses in the vagina. Following vaginal immunization, however, neither local nor distant IgA responses were detected. These results suggest that vaccines intended for protection of colonic-rectal and vaginal mucosal surfaces might best be administered by the rectal route.

Animals↗
PubMed1994

Proteolytic processing of reovirus is required for adherence to intestinal M cells.

Reovirus adheres specifically to apical membranes of mouse intestinal M cells and exploits M-cell transepithelial transport activity to enter Peyer's patch mucosa, where replication occurs. Proteolytic conversion of native reovirus to intermediate subviral particles (ISVPs) occurs in the intestine, but it is not known whether conversion is essential for interaction of virus with M cells. We tested the capacity of native virions, ISVPs, and cores (that lack outer capsid proteins) to bind to intestinal epithelial cells in vivo and found that only ISVPs adhered to M cells. Thus, intraluminal conversion of native reovirus to ISVPs is a prerequisite for M-cell adherence, and outer capsid proteins unique to ISVPs (either sigma 1 or products of mu 1) mediate interaction of virus with M-cell apical membranes.

Animals↗
PubMed1994

Regional differences in glycoconjugates of intestinal M cells in mice: potential targets for mucosal vaccines.

We have used a panel of lectins and antibodies to describe the composition of complex carbohydrates associated with M cells in various regions of the intestinal tract of adult BALB/c mice. The fucose-specific lectin Ulex europaeus agglutinin type I (UEA I) is a marker of M cells in the small intestine and recognized M cells at an early stage of differentiation. Subpopulations of M cells in a single follicle-associated epithelium (FAE) could be distinguished by different fucose-specific probes. Certain lectins revealed that M cells have basal processes that extend into the underlying lymphoid tissue. Colonic and rectal M cells display glycosylation patterns distinct from M cells of Peyer's patches and are characterized by terminal galactose. UEA I selectively adhered to Peyer's patch M cells in mucosal explants and in ligated intestinal loops in vivo. The lectin was taken up into endocytic vesicles and transported to the intra-epithelial pocket and other domains of the basolateral membrane. Thus M cell-specific glycoconjugates could serve as "receptors" for targeting of lectin-antigen conjugates to the mucosal immune system.

Animals↗
PubMed1994

Identification of R-GRAMP, a membrane glycoprotein of regulated secretory granules in primate cells.

Investigation of membrane assembly and traffic in the regulated secretory pathway may be facilitated by identification of membrane components that are unique to regulated secretory granules. To identify such markers, we isolated integral membrane proteins by Triton X-114 extraction from well-differentiated monolayers of an exocrine cell line, the goblet cell subclone (18N2) of the human colon carcinoma cell line HT29, and used the extracts as immunogens to produce monoclonal antibodies (mAbs). Immunofluorescence microscopy of HT29 goblet cell monolayers identified one mAb (MG-1) that labeled a component of mucin granule membranes. Immunofluorescence of frozen semithin sections of normal intestine, and various other human and monkey tissues, showed that this antigen is present in regulated secretory granule membranes of primate exocrine cells, endocrine cells, and tissue granulocytes. EM immunogold labeling of goblet cells, enteroendocrine cells and eosinophils confirmed that the antigen is associated with secretory granule membranes and not with plasma membranes. The antigen was identified by SDS-PAGE autoradiography of immunoprecipitates from HT29 goblet cells metabolically labeled with [35S]methionine and [35S]cysteine or [3H]glucosamine, as a glycoprotein with an apparent molecular mass ranging from 23 to 37 kDa. Digestion of immunoprecipitates with N-glycosidase F reduced the apparent mass to 16 to 19 kDa. This small, highly-glycosylated protein was named "R-GRAMP" (for regulated granule-associated membrane protein) to reflect its wide distribution in secretory granule membranes of regulated exocrine, endocrine and granulocytic cell types. This distribution suggests that it may play a common functional role in regulated secretion.

APUD Cells↗
PubMed1994

Mucosal immunization via M cells for production of protective secretory IgA antibodies.

Transepithelial transport of antigens by M cells in the epithelium associated with lymphoid follicles in the intestine delivers immunogens directly to organized mucosal lymphoid tissues, the inductive sites for mucosal immune responses. We have exploited M cell transport to generate and characterize specific monoclonal IgA antibodies that can prevent the interaction of pathogens with epithelial surfaces. The relative protective capacities of specific monoclonal IgA antibodies have been tested in vivo by generation of hybridoma tumors that result in the secretion of monoclonal IgA into the intestine. Using this method, we have established that the secretion of IgA antibodies recognizing a single surface epitope on enteric pathogens can provide protection against colonization or invasion of the intestinal mucosa.

Animals↗
PubMed1993

Transcytosis of the polymeric Ig receptor requires phosphorylation of serine 664 in the absence but not the presence of dimeric IgA.

MDCK cells expressing the polymeric immunoglobulin (poly-Ig) receptor, cocultured with IgA-producing hybridoma cells, transported dimeric IgA (dIgA) from the basolateral into the lumenal compartment, where it was recovered as secretory component-dIgA complexes. The tail of the receptor was phosphorylated on serines 664 and 726. Each serine was mutated to alanine. Appearance of A726 receptor at the basolateral surface was reduced approximately 5-fold. This was accompanied by a approximately 5-fold reduction in dIgA transcytosis. Basolateral delivery of receptor was not affected by mutation A664, and in the absence of dIgA, the receptor accumulated in recycling basolateral endosomes. In coculture, however, dIgA transcytosis by A664 receptor was normal. Thus, entry of receptor into the transcytotic pathway requires Ser-664 phosphorylation only in the absence of dIgA.

Amino Acid Sequence↗
PubMed1993

Monoclonal immunoglobulin A antibodies directed against cholera toxin prevent the toxin-induced chloride secretory response and block toxin binding to intestinal epithelial cells in vitro.

Secretory immunoglobulin A (IgA) antibodies directed against cholera toxin (CT) are thought to be important in resistance to oral challenge with virulent Vibrio cholerae, although alternative mechanisms for protection of intestinal epithelia against CT-induced fluid secretion have been proposed. The ability of anti-CT IgA to block the effects of CT on human enterocytes has not been directly tested because of the lack of a well-defined in vitro intestinal epithelial cell system to directly measure toxin action and the limited availability of purified anti-CT IgA antibodies. We have generated hybridomas that produce monoclonal IgA and IgG antibodies directed against CT by fusion of Peyer's patch cells with mouse myeloma cells after oral-systemic immunization of mice with CT and CT B-subunit protein. All of the anti-CT antibodies recognized the B subunit. Three clones (designated anti-CTB IgA-1, IgA-2, and IgA-3) which produced IgA antibodies in dimeric and polymeric forms were selected. Checkerboard immunoblotting demonstrated that IgA-1 recognized an epitope distinct from that recognized by IgA-2 and IgA-3 and that none of the antibodies were directed against the binding site of GM1, the intestinal cell membrane toxin receptor. The protective capacity of these IgAs was tested in vitro with human T84 colon carcinoma cells grown on permeable supports as confluent monolayers of polarized enterocytes. When each anti-CTB IgA was mixed with 10 nM CT and applied to the apical surfaces of T84 cell monolayers, all three IgAs blocked CT-induced Cl- secretion in a dose-dependent manner and completely inhibited binding of rhodamine-labelled CT to apical cell membranes. Thus, monoclonal anti-CTB IgA antibodies are sufficient to protect human enterocytes in vitro against CT binding and action.

Animals↗
PubMed1993

Analysis of the roles of antilipopolysaccharide and anti-cholera toxin immunoglobulin A (IgA) antibodies in protection against Vibrio cholerae and cholera toxin by use of monoclonal IgA antibodies in vivo.

Secretory immunoglobulin A (IgA) antibodies (sIgA) directed against cholera toxin (CT) and surface components of Vibrio cholerae are associated with protection against cholera, but the relative importance of specific sIgAs in protection is unknown. A monoclonal IgA directed against the V. cholerae lipopolysaccharide (LPS), secreted into the intestines of neonatal mice bearing hybridoma tumors, was previously shown to provide protection against a lethal oral dose of 10(7) V. cholerae cells. We show here that a single oral dose of 5 to 50 micrograms of the monoclonal anti-LPS IgA, given within 2 h before V. cholerae challenge, protected neonatal mice against challenge. In contrast, an oral dose of 80 micrograms of monoclonal IgA directed against CT B subunit (CTB) failed to protect against V. cholerae challenge. A total of 80 micrograms of monoclonal anti-CTB IgA given orally protected neonatal mice from a lethal (5-micrograms) oral dose of CT. Secretion of the same anti-CTB IgA antibodies into the intestines of mice bearing IgA hybridoma backpack tumors, however, failed to protect against lethal oral doses of either CT (5 micrograms) or V. cholerae (10(7) cells). Furthermore, monoclonal anti-CTB IgA, either delivered orally or secreted onto mucosal surfaces in mice bearing hybridoma tumors, did not significantly enhance protection over that provided by oral anti-LPS IgA alone. These results demonstrate that anti-LPS sIgA is much more effective than anti-CT IgA in prevention of V. cholerae-induced diarrheal disease.

Administration, Oral↗
PubMed1993

The role of transepithelial transport by M cells in microbial invasion and host defense.

Transepithelial transport of antigens by M cells in the epithelium associated with lymphoid follicles in the intestine delivers immunogens directly to organized mucosal lymphoid tissues, the inductive sites for mucosal immune responses. We have exploited M cell transport to generate and characterize specific monoclonal IgA antibodies that can prevent interaction of pathogens with epithelial surfaces. The relative protective capacities of specific monoclonal IgA antibodies have been tested in vivo by generation of hybridoma tumors that result in secretion of monoclonal IgA into the intestine. Using this method, we have established that secretion of IgA antibodies recognizing a single surface epitope on enteric pathogens can provide protection against colonization or invasion of the intestinal mucosa.

Animals↗
PubMed1992

Transepithelial transport and mucosal defence II: secretion of IgA.

In this second article on mucosal defence and transepithelial transport, Jean-Pierre Kraehenbuhl and Marian Neutra discuss the part played by a special class of antibody, polymeric IgA, in the protection of mucosal surfaces lining the digestive, respiratory and genital tracts, and the implications for mucosal vaccines. Polymeric IgA crosslinks luminal antigens or pathogens, thus preventing their interaction with epithelial cells. Following stimulation by antigen in the organized mucosal lymphoid tissue, effector B lymphocytes enter the circulation and migrate to distant mucosal or glandular sites, where they differentiate into polymeric-IgA-producing plasma cells. These antibodies reach the environment by transport across the epithelial cells of mucosal and glandular tissues.

Journal Article↗
PubMed1992

Transepithelial transport and mucosal defence I: the role of M cells.

How do cells of the immune system encounter the majority of antigens that enter the body through the gut and airways? The epithelia lining these systems contain a remarkable cell type, the M cell, that delivers antigens across the epithelium to lymphocytes and macrophages. In this article, Marian Neutra and Jean-Pierre Kraehenbuhl describe the structure of the M cell, its function in promoting the immune response and its exploitation by invading pathogens. In the next issue of Trends in Cell Biology, these authors will review the other immunological function of epithelia, secretion of polymeric IgA.

Journal Article↗
PubMed1992

Mechanism of cholera toxin action on a polarized human intestinal epithelial cell line: role of vesicular traffic.

The massive secretion of salt and water in cholera-induced diarrhea involves binding of cholera toxin (CT) to ganglioside GM1 in the apical membrane of intestinal epithelial cells, translocation of the enzymatically active A1-peptide across the membrane, and subsequent activation of adenylate cyclase located on the cytoplasmic surface of the basolateral membrane. Studies on nonpolarized cells show that CT is internalized by receptor-mediated endocytosis, and that the A1-subunit may remain membrane associated. To test the hypothesis that toxin action in polarized cells may involve intracellular movement of toxin-containing membranes, monolayers of the polarized intestinal epithelial cell line T84 were mounted in modified Ussing chambers and the response to CT was examined. Apical CT at 37 degrees C elicited a short circuit current (Isc: 48 +/- 2.1 microA/cm2; half-maximal effective dose, ED50 integral of 0.5 nM) after a lag of 33 +/- 2 min which bidirectional 22Na+ and 36Cl- flux studies showed to be due to electrogenic Cl- secretion. The time course of the CT-induced Isc response paralleled the time course of cAMP generation. The dose response to basolateral toxin at 37 degrees C was identical to that of apical CT but lag times (24 +/- 2 min) and initial rates were significantly less. At 20 degrees C, the Isc response to apical CT was more strongly inhibited (30-50%) than the response to basolateral CT, even though translocation occurred in both cases as evidenced by the formation of A1-peptide. A functional rhodamine-labeled CT-analogue applied apically or basolaterally at 20 degrees C was visualized only within endocytic vesicles close to apical or basolateral membranes, whereas movement into deeper apical structures was detected at 37 degrees C. At 15 degrees C, in contrast, reduction to the A1-peptide was completely inhibited and both apical and basolateral CT failed to stimulate Isc although Isc responses to 1 nM vasoactive intestinal peptide, 10 microM forskolin, and 3 mM 8Br-cAMP were intact. Re-warming above 32 degrees C restored CT-induced Isc. Preincubating monolayers for 30 min at 37 degrees C before cooling to 15 degrees C overcame the temperature block of basolateral CT but the response to apical toxin remained completely inhibited. These results identify a temperature-sensitive step essential to apical toxin action on polarized epithelial cells. We suggest that this event involves vesicular transport of toxin-containing membranes beyond the apical endosomal compartment.

Biological Transport↗
PubMed1992

Monoclonal secretory immunoglobulin A protects mice against oral challenge with the invasive pathogen Salmonella typhimurium.

Hybridomas producing monoclonal immunoglobulin A (IgA) antibodies against Salmonella typhimurium were generated by mucosal immunization of BALB/c mice with attenuated strains of S. typhimurium and subsequent fusion of Peyer's patch lymphoblasts with myeloma cells. To test the role of secretory IgA (sIgA) in protection against Salmonella sp., we analyzed in detail the protective capacity of a monoclonal IgA, Sal4, produced in polymeric as well as monomeric forms, that is directed against a carbohydrate epitope exposed on the surface of S. typhimurium. BALB/c mice bearing subcutaneous Sal4 hybridoma tumors and secreting monoclonal sIgA into their gastrointestinal tracts were protected against oral challenge with S. typhimurium. This protection was directly dependent on specific recognition by the monoclonal IgA, since mice secreting Sal4 IgA from hybridoma tumors were not protected against a fully virulent mutant that lacks the Sal4 epitope. Although monoclonal Sal4 IgA was present in the bloodstreams and tissues of tumor-bearing mice, it did not protect against intraperitoneal challenge and did not possess complement-fixing or bacteriocidal activity in vitro. Taken together, these results indicate that secretion of sIgA alone can prevent infection by an invasive enteric pathogen, presumably by immune exclusion at the mucosal surface.

Animals↗