Cell vacuolation caused by Vibrio cholerae hemolysin.

Non-O1 strains of Vibrio cholerae implicated in gastroenteritis and diarrhea generally lack virulence determinants such as cholera toxin that are characteristic of epidemic strains; the factors that contribute to their virulence are not understood. Here we report that at least one-third of diarrhea-associated nonepidemic V. cholerae strains from Mexico cause vacuolation of cultured Vero cells. Detailed analyses indicated that this vacuolation was related to that caused by aerolysin, a pore-forming toxin of Aeromonas; it involved primarily the endoplasmic reticulum at early times (approximately 1 to 4 h after exposure), and resulted in formation of large, acidic, endosome-like multivesicular vacuoles (probably autophagosomes) only at late times (approximately 16 h). In contrast to vacuolation caused by Helicobacter pylori VacA protein, that induced by V. cholerae was exacerbated by agents that block vacuolar proton pumping but not by endosome-targeted weak bases. It caused centripetal redistribution of endosomes, reflecting cytoplasmic alkalinization. The gene for V. cholerae vacuolating activity was cloned and was found to correspond to hlyA, the structural gene for hemolysin. HlyA protein is a pore-forming toxin that causes ion leakage and, ultimately, eukaryotic cell lysis. Thus, a distinct form of cell vacuolation precedes cytolysis at low doses of hemolysin. We propose that this vacuolation, in itself, contributes to the virulence of V. cholerae strains, perhaps by perturbing intracellular membrane trafficking or ion exchange in target cells and thereby affecting local intestinal inflammatory or other defense responses.

Evidence for cytotoxic T lymphocyte response against human lung cancer: reconstitution of antigenic epitope with peptide eluted from lung adenocarcinoma MHC class I.

BACKGROUND: Cancer-associated, major histocompatibility complex (MHC)-restricted peptide antigens have been elucidated in human melanomas and ovarian, breast, and renal carcinomas; but relatively little is known about lung cancer antigens. METHODS: To work toward delineation of lung cancer-associated antigens, we developed tumor infiltrating lymphocytes (TILs), peripheral blood mononuclear cell-derived cytolytic T cell lines (CTL), autologous lung cancer cell lines, and normal lung cell lines from 17 patients undergoing lung cancer resections. The TILs and CTL lines were subsequently evaluated for markers of activation and specific lysis of autologous or allogeneic lung cancer cell lines or both. RESULTS: Freshly isolated TILs contained a more activated T cell population compared with the patients' peripheral blood T cells as evidenced by an increased expression of HLA-DR, CD25, and CD45RO. TILs isolated from 15 patients lysed allogeneic lung cancer lines. TILs lysed autologous lung cancer but not autologous normal lung or Epstein-Barr virus transformed B cell lines (B-LCL) in 4 of 8 cases tested, suggesting tumor specificity. A CTL line (RHPBL57.1) was generated from peripheral blood mononuclear cells of an HLA-A24(+) patient by stimulation against an established HLA-A24(+) allogeneic lung cancer cell line. RHPBL57.1 lysed the lung cancer cell line in an HLA-A24-restricted manner. Moreover, RHPBL57.1 specifically lysed autologous B-LCL pulsed with peptides, eluted from MHC class I and isolated from the HLA-A24(+) lung cancer cell line. CONCLUSIONS: TILs isolated from patients with lung cancer are predominantly an activated population of T cells with evidence of tumor and MHC class I-restricted lysis. Furthermore, we provide evidence for a lung cancer-associated, MHC class I-bound peptide antigen(s) that reconstitutes the epitope recognized by a lung cancer specific CD8(+) T cell line derived from a patient with lung cancer.

Intragastric immunization of rats with Entamoeba histolytica trophozoites induces cecal mucosal IgE, eosinophilic infiltration, and type I hypersensitivity.

We have investigated the role of IgE in the local immunity of intestinal amebiasis, a parasitic infection known to induce specific antibody-forming cells (AFC) and IgA antibodies in rodents and humans. We found that intragastric immunization of rats with glutaraldehyde-fixed Entamoeba histolytica trophozoites significantly increased antiameba AFC in the Peyer's patches and spleen and that the lamina propria of the cecum from immunized animals was infiltrated by eosinophils armed with IgE antibodies. Morphometric analysis showed that IgE-containing cells and eosinophils were nearly three times more abundant in the cecum of immunized rats. Antigenic challenge with amebal lysates provoked an increase in the short-circuit current and in the transepithelial potential difference in Ussing-chambered cecum preparations from immunized rats. Although eosinophilia and the increase of IgE are common consequences of infection by parasitic worms, our results indicate that local immunity in intestinal amebiasis also involves IgE deposition, eosinophil infiltration, and type I hypersensitivity, which may explain some symptoms of amebic dysentery such as colic, abdominal tension, tenesmus, and bloody stools.

Immunodominant Entamoeba histolytica antigens recognized by serum and intestinal antibodies after local or systemic immunization of mice with glutaraldehyde fixed trophozoites.

We performed an immunoblot analysis of the main E. histolytica proteins recognized by immune sera and intestinal fluids of Balb/c mice immunized with glutaraldehyde fixed trophozoites (GFT) by intragastric, rectal and intraperitoneal routes, to determine if there were differences in the amebic antigens immunodominantly recognized at mucosal and systemic levels. The antigen patterns recognized by mice immunized via intraperitoneal and rectal routes were complex and similar suggesting that the immunization route (systemic or local), does not influence the recognition pattern elicited at mucosal or systemic levels. However, the number of amebic bands recognized after intragastric immunization was very low. The molecular weights of the principal amebic proteins recognized by serum antibodies were 150-130, 116, 104, 84, 56, 42, 18, and 16 kDa. The intestinal fluids of mice immunized via intraperitoneal and rectal routes contained antibodies that recognized five bands of 220-200, 150-134, 93-84, 43-41, and 16-14 kDa. These results suggest that there are differences in the number of immunodominant amebic antigens recognized at mucosal and systemic levels. Moreover we found that the bands of 150, 39 and 19 kDa. were mainly recognized by IgG, whereas the bands of 116, 93, and 16 were mainly recognized by IgM, indicating differences between the antigens immunodominantly recognized by serum antibodies from different isotypes.

Quantification and isotype analysis of the serum anti-amebic antibody response produced after mucosal and systemic immunization in male and female mice.

It has not been established whether seric antiamebic antibodies are produced after local presence of amebas, or only after they invade the tissues. Therefore, the purpose of this work was to determine if mucosal immunization with glutaraldehyde-fixed trophozoites (GFT) could induce seric anti-amebic antibody responses in mice. We determined by ELISA the quality and quantity of anti-E. histolytica seric antibody responses induced after either a single or multiple doses of GFT by intragastric, rectal and intraperitoneal routes. Seric anti-amebic antibody responses were elicited by both mucosal or systemic immunization although such responses induced via intragastric route were low. However, the responses achieved via rectal route were high, suggesting that the local presence of amebas without mucosal tissue damage is enough to stimulate seric anti-amebic response. The highest seric anti-amebic responses were obtained via intraperitoneal route. The antibody isotype of the induced responses was influenced by the immunization route. The major isotypes produced via intraperitoneal and rectal routes were IgM and IgG. The predominant isotype elicited by intragastric route was IgM whereas the IgA and IgG responses were small and of the same magnitude. The lowest IgA anti-amebic response was produced by intraperitoneal route. In addition, our data show that in general GFT immunization elicits higher anti-amebic antibody responses in female than in male mice.

Entamoeba histolytica: induction and isotype analysis of antibody producing cell responses in Peyer's patches and spleen after local and systemic immunization in male and female mice.

The purpose of this work was to determine if anti-amebic antibody producing cell responses could be elicited in Peyer's patches and spleen in mice locally or systemically immunized with glutaraldehyde-fixed trophozoites of Entamoeba histolytica (GFT). The animals were inoculated with either a single or four doses of GFT via intragastric, rectal, and intraperitoneal routes. The anti-amebic antibody producing cell responses were analyzed by a spot forming cell assay (ELISPOT). The kinetics of antibody response revealed that a single dose of GFT by any route evoked anti-amebic responses in Peyer's patches and spleen. Furthermore, antibody producing cells of the three major isotypes were produced in both Peyer's patches and spleen of the mice receiving four doses of GFT, by either local or systemic routes. Our results indicate that immunization with GFT can induce a considerable number of specific antibody producing cells, which seem to remain in the Peyer's patches. After rectal and intraperitoneal immunization, females produced higher anti-amebic responses than males. Since either local or systemic immunization with GFT elicits both mucosal and systemic anti-amebic responses, this strategy should be considered as a promising tool for future elaboration of an anti-amebic vaccine.

The use of an ELISPOT assay to evaluate intestinal and systemic antibody responses to locally administered Entamoeba histolytica antigen in mice.

The local induction of humoral immune response to Entamoeba histolytica trophozoites in the gut has not been studied. This work reports some of our recent studies aimed to induce optimal immune responses against E. histolytica in mice and to describe a novel approach for monitoring mucosal immune responses induced in the gastrointestinal tract and expressed locally and systemically. We have compared the kinetics of both mucosal and systemic primary antibody responses to E. histolytica in the Peyer's patches (PP) and the spleen in Balb/c mice after a single dose of glutaraldehyde fixed amebas (GFA) by intragastric (IG), rectal (R), and intraperitoneal (IP) routes. The number of antibody-secreting cells directed to E. histolytica was assessed by the technique of ELISPOT on nitrocellulose filters. The antibody response to E. histolytica was detected in both PP and spleen with the three routes, indicating that either mucosal or systemic stimulation by GFA generates both types of response. We also determined the total antibody response in intestinal fluids and the antibody secretions from spleen and PP cells in vitro and found differences between male and female mice.

Sex differences in systemic and local immune responses to Entamoeba histolytica after intraperitoneal and rectal immunization in Balb/c mice.

The antibody responses against Entamoeba histolytica in Peyer's patches and spleen after rectal, intraperitoneal and intragastric immunization with glutaraldehyde-fixed amebas (GFA) was compared between male and female mice by the use of a spot forming cell (SFC) assay. We found that female mice elicit significant higher anti-amebic SFC responses than male mice, in both spleen and Peyer's patches after rectal and intraperitoneal stimulation, whereas by intragastric route females show higher responses than males in spleen but not in Peyer's patches.

Kinetics of the anti-amebic antibody producing cells response in Peyer's patches and spleen after both local and systemic stimulation in Balb/c mice.

The kinetics of the anti-amebic antibody producing cell (APC) response was analyzed by a spot forming cell (SFC) assay, after a single dose of glutaraldehyde fixed amebas (GFA) by intragastric, rectal and intraperitoneal routes. It was found that mice elicited both local and systemic immune responses in Peyer's patches and spleen, by either local or systemic stimulation. The analysis of the isotype of the anti-amebic APC in mice given four times the GFA by the mentioned routes revealed that mice produced anti-amebic APC of the three major isotypes in both spleen and Peyer's patches.

Suppression of follicular trapping of antigen-antibody complexes in mice treated with anti-IgM or anti-IgD antibodies from birth.

Mice were treated from birth with either goat anti-mouse IgM or with a monoclonal anti-IgD antibody. When they were 8 weeks old, cohorts of these mice were given 125I-labelled antigen, either by itself, or in an antigen-antibody complex. Anti-IgM-treated mice, which did not develop follicular structures in their spleens, failed to retain immune complexes on follicular dendritic cells in the characteristic pattern. Anti-IgD-treated mice, which had small follicles consisting of IgM+ IgD- B cells in their spleens, retained substantially smaller amounts of immune complexes than normal. These results support the concept that B lymphocytes transport antigen-antibody complexes to follicular dendritic cells. Furthermore, in the mouse it seems likely that this is mediated by both IgM+ IgD+ and IgM+ IgD- B cells.

Differing effects of monoclonal anti-hapten antibodies on humoral responses to soluble or particulate antigens.

This study compares the effects of passive administration of monoclonal anti-hapten (DNP) antibodies on primary plaque-forming cell (PFC) responses in mice to either soluble (DNP-keyhole limpet haemocyanin [KLH] ) or particulate (TNP-erythrocyte) antigens. IgM, IgG1, IgG2a and IgG2b antibodies at doses up to 500 micrograms induced at best a modest suppression of the IgM response, and reproducibly enhanced the IgG response to DNP-KLH by up to 30-fold. In contrast, with the particulate antigen only the IgM antibody enhanced IgG PFC; IgG2 antibodies, and one out of two IgG1 antibodies caused marked suppression of the primary response to TNP-RBC. This required antibody with an intact Fc portion. The enhancement of IgG responses to soluble antigen presumably reflects rapid B cell priming by immune complexes trapped by follicular dendritic cells in lymphoid follicles, in agreement with earlier data. These results indicate that the nature of the antigen can markedly influence the immunoregulatory effects of antibodies on humoral responses.

Follicular trapping of hapten-erythrocyte-antibody complexes in mouse spleen.

In this study we followed the in vivo fate and distribution of hapten-coupled sheep red blood cells (TNP-SRBC) coated with 125I-labelled anti-hapten antibody in mice. The majority of these complexes was rapidly taken up by the liver, and by macrophages in the marginal zone of the spleen. Within a few hours, however, marginal zone labelling diminished and label appeared in the corona of lymphoid follicles. After 1-7 days the only remaining label was associated with areas of follicular dendritic cells. This follicular trapping was abolished by decomplementing mice with cobra venom factor. These results, therefore, demonstrate that immune complexes made with a large, particulate antigen are retained for long periods in lymphoid follicles in an analogous fashion to soluble complexes. We assume (but have not proved) that the complexes retained by dendritic cells contain soluble erythrocyte antigens released from macrophages, rather than intact RBC. This possibility is strengthened by the presence of significant amounts of free, functional antibody in the circulation of mice given TNP-SRBC coated with antibody in vitro. The possible significance of immune complex trapping in antibody-induced suppression of anti-RBC responses is discussed.