Sterilizing immunity against experimental Helicobacter pylori infection is challenge-strain dependent.

The development of a murine model of Helicobacter pylori infection through serial in vivo passage of candidate strains has enabled a quantitative assessment of vaccine efficacy. In this study we compare infection with and protection against challenge from both CagA(+) type I, and CagA(-) type II in vivo adapted isolates. In vivo passage of a type II H. pylori isolate resulted in a highly infectious strain (X47-2AL), capable of reproducibly infecting mice to high density (10(7) CFU/g of gastric tissue). Similarly adapted type I strains were found to colonize mice at a significantly lower level (10(4)-10(5) CFU/g tissue). Mucosal immunization with recombinant urease (rUre) significantly protected animals against both types. Protection against X47-2AL was characterized by a > or =100-fold (or 2 log) reduction in bacterial density. However, the presence of a residual infection highlighted the inability to achieve sterilizing immunity against this strain. The level of protection appeared independent of challenge dose, and was stable for up to 6 months, all animals exhibiting a low-level residual infection that did not recrudesce with time. Similarly immunized mice challenged with isolates representing the residual infection were also protected, confirming that they did not represent a sub-population of H. pylori that could escape immunity. Immunization and challenge studies with type I adapted-isolates, demonstrated a similar 2-3 log reduction in the bacterial burden, but that in this instance resulted in sterilizing immunity. These results suggest varied specificity for the murine host by different Helicobacter strains that can influence the outcome of both infection and immunity.

Oral immunization with recombinant Helicobacter pylori urease confers long-lasting immunity against Helicobacter felis infection.

Recombinant Helicobacter pylori urease (rUre) has been shown to confer protection against challenge with Helicobacter felis in mice. The purpose of the present study was to examine duration of the immune response and long-term protective efficacy of immunization with rUre. Swiss Webster mice were orally immunized four times at weekly intervals with 100 microg rUre plus 5 microg heat-labile enterotoxin of Escherichia coli (LT) adjuvant, or with LT only. At 4, 10, 20 or 40 weeks post immunization, 25 rUre-immunized mice and control mice were challenged with H. felis and sacrificed at 2 or 10 weeks post-challenge. H. felis infection was assessed by gastric urease assay and by histology. Anti-H. pylori urease specific antibody levels were measured in serum and saliva both pre- and post-challenge. Over the 40 week time period, the infection rates in rUre-immunized mice were significantly lower than those in controls (p < 0.05) as assessed by gastric urease activity. Protection ranged from 79 100% at 2 weeks post-challenge and 63-78% at 10 weeks post-challenge. Gastric bacterial density in rUre-immunized mice was significantly lower than that of controls (p < 0.03) as determined by histologic assessment. Anti-urease antibody levels remained elevated in the serum and mucosal compartments at 39 weeks following immunization. This study shows that immunization with rUre plus LT results in long-lasting protective immunity against challenge with H. felis.

Immunization of mice with urease vaccine affords protection against Helicobacter pylori infection in the absence of antibodies and is mediated by MHC class II-restricted responses.

We examined the roles of cell- and antibody-mediated immunity in urease vaccine-induced protection against Helicobacter pylori infection. Normal and knockout mice deficient in major histocompatibility complex (MHC) class I, MHC class II, or B cell responses were mucosally immunized with urease plus Escherichia coli heat-labile enterotoxin (LT), or parenterally immunized with urease plus aluminum hydroxide or a glycolipid adjuvant, challenged with H. pylori strain X47-2AL, and H. pylori organisms and leukocyte infiltration in the gastric mucosa quantified. In an adjuvant/route study in normal mice, there was a direct correlation between the level of protection and the density of T cells recruited to the gastric mucosa. In knockout studies, oral immunization with urease plus LT protected MHC class I knockout mice [beta2-microglobulin (-/-)] but not MHC class II knockout mice [I-Ab (-/-)]. In B cell knockout mice [microMT (-/-)], vaccine-induced protection was equivalent to that observed in immunized wild-type (+/+) mice; no IgA+ cells were detected in the stomach, but levels of CD4(+) cells equivalent to those in the wild-type strain (+/+) were seen. These studies indicate that protection of mice against H. pylori infection by immunization with the urease antigen is dependent on MHC class II-restricted, cell-mediated mechanisms, and antibody responses to urease are not required for protection.

Rectal and intranasal immunizations with recombinant urease induce distinct local and serum immune responses in mice and protect against Helicobacter pylori infection.

To determine the optimal inductive sites for immunization against Helicobacter pylori infection, the protective efficacy of recombinant urease (rUre) was assessed for mice given the vaccine by either the oral (p.o.), intranasal (i.n.), or rectal route. When mice were immunized with rUre (25 microg p.o. or rectally or 10 microg i.n.) plus heat-labile toxin from Escherichia coli as the mucosal adjuvant, all routes afforded protection against challenge with H. pylori, as indicated by a significant reduction in gastric urease activity (P < 0.0005) compared to that of sham-immunized controls. Quantitative H. pylori culture of stomach tissue demonstrated a >97% reduction in bacterial burden in mice immunized by all routes (P < 0.05). Induction of antiurease immunoglobulin A (IgA) levels in gastric luminal secretions after p.o. immunization was greater than after i.n. administration (means, 6.0 and 1.02 ng/ml, respectively) and was dependent upon challenge with H. pylori. However, immunization by the rectal route resulted in the generation of the highest levels of gastric antiurease IgA (mean, 40. 89 ng/ml), which was detectable prior to challenge with H. pylori. Immunohistochemical staining of stomach tissue for cells secreting urease-specific antibody and CD4(+) T cells showed levels of recruitment to be dependent upon challenge with H. pylori and equivalent for all routes. These results identify both the rectum and nasal passages as suitable inductive sites for urease immunization.

Gastritis in urease-immunized mice after Helicobacter felis challenge may be due to residual bacteria.

BACKGROUND & AIMS: Oral immunization with recombinant Helicobacter pylori urease (rUre) coadministered with a mucosal adjuvant protects mice against challenge with Helicobacter felis. In this study, the duration of protection and gastritis after challenge were characterized at sequential time intervals up to 1 year. METHODS: Outbred Swiss-Webster mice were orally immunized with rUre plus adjuvant and examined for the presence of H. felis infection and leukocyte infiltration into the gastric mucosa. RESULTS: When defined by gastric urease activity, 70%-95% of rUre-immunized mice were protected for between 2 and 57 weeks. Challenge with H. felis increased the inflammatory response in the gastric mucosa of rUre-immunized mice, which also had elevated CD4+ and CD8+ T cells. The CD8+ cells represented a population of gastric intraepithelial cells, which expressed the mucosal alpha E-integrin. Epithelial changes consisting of parietal cell loss and hyperplasia of the epithelium occurred in approximately 20% of the mice. Antimicrobial triple therapy significantly decreased the degree of gastritis and epithelial alteration in the stomach. CONCLUSIONS: These results indicate that oral immunization of mice with rUre produces a long-lasting inhibition of H. felis infection but that residual bacteria may produce a persistent lymphocytic infiltration under these experimental conditions.

Oral immunization with recombinant Helicobacter pylori urease induces secretory IgA antibodies and protects mice from challenge with Helicobacter felis.

Helicobacter pylori, a gram-negative spiral bacterium, is the cause of chronic superficial (type B) gastritis and peptic ulcer disease. The urease enzyme of H. pylori was expressed as an inactive recombinant protein in Escherichia coli, purified as particulate structures of 550-600 kDa molecular mass with a diameter of approximately 12 nm. Given orally, 5 micrograms of urease with an appropriate mucosal adjuvant, such as the labile toxin of E. coli, protected 60%-100% of mice against challenge with virulent Helicobacter felis. Protection correlated with the level of secretory IgA antibodies against urease. Oral administration of antigen was as effective or better than intragastric administration. Parenteral injection of antigen or intragastric administration of high-dose antigen without adjuvant elicited serum IgG but no IgA antibodies and did not confer protection. Recombinant urease as an oral vaccine candidate deserves further investigation as an approach to the prevention of Helicobacter-induced chronic gastroduodenal diseases in humans.

Uptake and transport of copolymer biodegradable microspheres by rabbit Peyer's patch M cells.

In this study, we demonstrate the role of M cells in uptake of poly(D-L-lactic-co-glycolic acid) (PLGA) microspheres and transport into rabbit Peyer's patches. Microspheres 1 to 10 microns in diameter composed of 50:50 lactic acid:glycolic acid were instilled into intestinal segments containing jejunal or ileal Peyer's patches, and uptake by M cells was examined by electron microscopy. PLGA microspheres visualized as electron-lucent, spherical particles were taken up by M cells by pseudopod-like extensions of the M cell apical membrane and translocated to the pocket region containing mononuclear leukocytes within 60 min. These results indicate that PLGA microspheres can be directed to M cell apical surfaces for delivery to immunocompetent cells in gut-associated lymphoid tissues.

Induction of autoimmune disease in mice by germline alteration of the T cell receptor gene expression.

Germline expression of rearranged TCR alpha-chain transgenes with the Ig H chain enhancer reproducibly elicits T cell-mediated autoimmune disease in the thyroid gland, gastric mucosa, Langerhans islets, salivary gland, ovaries, and testes in selected strains of normal mice. Multiple organs are destroyed in a single transgenic mouse and the same organ in transgenic strains with different MHC background, suggesting the transgene expression can elicit self-reactive T cell clones having different Ag specificities and MHC restrictions. Construction of this autoimmune-inducing TCR alpha EH transgene does not require particular V alpha J alpha gene segments or Ag specificities. Moreover, the autoimmune disease can be adoptively transferred to syngeneic normal mice by T cells expressing endogenous TCR alpha-chains. Taken together, these results indicate that the TCR alpha EH transgene expression does not suppress endogenous alpha-chain gene rearrangement and may trigger the expansion/activation of various self-reactive T cells expressing endogenous TCR alpha- and beta-chains. Furthermore, it appears that the transgene-induced autoimmune T cells are not deleted in the normal thymus or rendered anergic upon contact with the normal target self Ag, but can be controlled by a T cell-dependent mechanism, since transfer of the transgenic bone marrow cells to histocompatible SCID mice produces the same autoimmune disease as in the donors, and the autoimmune development in the SCID mice is effectively prevented by co-transfer of syngeneic nontransgenic T cells. This novel autoimmune model produced by genetic manipulation of the T cell lineage, not the target self Ag or the environment of T cell differentiation/selection, should be useful for elucidating the immunologic and genetic basis of autoimmune disease.

Lymphocyte compartments in antigen-sampling regions of rabbit mucosal lymphoid organs.

Uptake and delivery of antigens to immunocompetent cells in the gut are critical factors for the development of oral vaccines. Particulate antigens are transported within minutes by M cells to intraepithelial lymphocytes and into the follicle dome. The dome contains B cells, CD4+ T cells, and macrophages, indicating that the cells involved in antigen presentation are located below the dome's epithelium. The high number of M cells in rabbits and the development of monoclonal antibodies against rabbit lymphocytes have enabled the detailed study of lymphocytes associated with M cells. The follicle epithelium of rabbit Peyer's patches contains B cells and a population of CD4-/CD8-, major histocompatibility complex class II+ mononuclear cells of unknown function. These cells are phenotypically distinct from T cells in follicle domes, in T cell-dependent areas, in villus epithelium, or in villus lamina propria. In addition, lymphocytes in M-cell pockets express an activation antigen (3B6) not found on CD4+ or CD8+ cells in T cell-dependent areas. These results indicate that M-cell pocket lymphocytes in follicle epithelium form a phenotypically distinct compartment situated at the interface between M-cell-driven antigen uptake and the mucosal immune system.

Correlation of extracellular matrix components with the cytoarchitecture of mouse Peyer's patches.

The distribution patterns of extracellular matrix elements were determined to ascertain whether they play a role in the localization of lymphocytes in discrete T-cell, B-cell and dome antigen-processing domains within Peyer's patches. Antibodies against collagen types I, III and IV, laminin and fibronectin were applied to cryosections of mouse Peyer's patches and localized by direct or indirect immunoperoxidase methods. T-cell domains were identified with a monoclonal antibody against Thy-1.2. Labeled reticular fibers in distinctive patterns were more numerous in parafollicular and dome areas than within follicles. Germinal centers contained few such fibers. In parafollicular areas, fibers were oriented predominantly toward follicle domes; their distribution corresponded to T-cell zones and lymphocyte traffic areas, with their orientation being parallel to the migration pathways of lymphocytes from high endothelial venules to the antigen-processing domes. Subepithelial and subendothelial basal laminae were immunopositive for type-IV collagen, laminin and fibronectin. The dome subepithelial basal lamina had pore-like discontinuities through which lymphocytes migrated to and from the epithelium. The correspondence of the distribution patterns of extracellular matrix to specific functional domains of Peyer's patches suggests that this matrix provides a structural framework for lymphocyte migration and localization.

Monoclonal antibody-directed targeting of fluorescent polystyrene microspheres to Peyer's patch M cells.

The ability to deliver particulates to Peyer's patch M cells for uptake into gut-associated lymphoid tissue was examined by administering simultaneously fluorescent green and red polystyrene microspheres into NZW rabbit intestinal loops containing Peyer's patches. Whereas green and red microspheres were taken up by M cells at equivalent concentrations (120 +/- 17 versus 125 +/- 18/mm length of dome), particles conjugated to the anti-M-cell monoclonal antibody 5B11 (IgM, kappa) were internalized by M cells 3-3.5 times more efficiently than conjugates displaying IgM of unrelated specificity (TEPC 183) or native particles of the reciprocal colour inoculated into the same loop at a comparable load. The microspheres formed a concentration gradient from lumen to subepithelial dome, and localized on M-cell apical membranes, M-cell pockets, and subepithelial domes. The transport rate across M cells of 5B11 or TEPC 183 conjugates was similar to that of untreated microspheres. These observations show that intestinal uptake into Peyer's patches can be upregulated by targeting M-cell luminal membrane structures.

Ultrastructural and cytoarchitectural features of lymphoreticular organs in the colon and rectum of adult BALB/c mice.

The structure and function of colonic mucosal lymphoid organs remain largely unexplored, especially in the rectum hidden within the pelvic vault. Two-month-old female BALB/c mice were anesthetized, and the entire colon was removed from cecum to anus. Distal colonic patches were then prepared for electron microscopy or were quick-frozen and sectioned for immunoperoxidase localization of B cells and T cell subsets. Aggregated lymphoid follicles were distributed irregularly along the entire colon with an average of 1.4 patches per centimeter of colon length. There were large collections of follicles opposite the ileocecal valve (cecal patches), variable numbers of patches throughout the colon, and at least one patch within 10 mm of the anus (rectal patch). Follicles were adjacent to branching crypts lined by epithelium infiltrated by lymphoid cells and containing few goblet cells. In electron micrographs, M cells were identified by their short, irregular microvilli; intraepithelial lymphoid cells; reduced lysosomal dense bodies; and an expanded tubulovesicular network. Small germinal centers were seen. Cytoarchitectural components of colonic lymphoid follicles and Peyer's patch follicles were remarkably similar, despite differences in surrounding mucosa and luminal microbial exposure. The presence of organized lymphoid tissue with M cells and germinal centers suggests that transepithelial particle transport and antigen recognition can take place in the rectum. Whether such tissue has the capacity for uptake of luminal microorganisms is of particular interest, not only because colonic follicles may be sites for local initiation of immune responses but also because they may be important entry points for systemic infection.

Phenotypically distinct subpopulations of T cells in domes and M-cell pockets of rabbit gut-associated lymphoid tissues.

Follicle epithelium and domes of gut-associated lymphoid tissue (GALT) contain populations of lymphocytes which first contact antigen taken up from the intestine. In order to study the association of lymphocytes with M cells in follicle epithelium, monoclonal antibodies (mAb) were generated by immunizing BALB/c mice with lymphocytes populating GALT domes from NZW rabbits, and their specificity was assessed by immunohistochemistry and flow cytometry. mAb 3C10 (IgM) and 3B6 (IgG3) recognized subpopulations of intraepithelial lymphocytes associated with M cells. mAb 3C10 also identified macrophage-lymphocyte clusters in domes and tangible body macrophages in germinal centres of GALT but did not react with cells in T-dependent areas (TDA) or B cells in follicles. mAb 3B6 recognized lymphocytes in domes and B cells in follicles but not T cells in TDA of GALT. The distribution of 3B6+ cells overlapped with, but was more restricted than, that of Ia+ cells. Analysis of lymphocytes in follicle epithelium showed that greater than 95% of lymphocytes associated with M cells were Ia+. T cells represented approximately 95% of intraepithelial lymphocytes in the appendix and approximately 65% in Peyer's patches. A majority of intraepithelial lymphocytes was recognized by mAb 3B6, but mAb 3C10 identified only approximately 30%. Because neither 3C10 nor 3B6 recognized lymphocytes in TDA of GALT, these results indicate that most lymphocytes associated with M cells are a distinct phenotype of Ia+ T cells.

F(ab')2 anti-CD4 and intact anti-CD4 monoclonal antibodies inhibit the accumulation of CD4+ T cells, CD8+ T cells, and B cells in the kidneys of lupus-prone NZB/NZW mice.

Murine lupus in NZB/NZW (B/W) mice is characterized by immune-complex glomerulonephritis and lymphocytic infiltration of several organs, including the kidney. We recently showed that treatment of B/W mice with F(ab')2 anti-CD4 monoclonal antibody retards autoimmunity by inhibiting the function of CD4+ cells and not by depleting them. To determine if treatment with F(ab')2 anti-CD4 prevented lymphocytic infiltration of kidneys or simply inhibited the function of the infiltrating lymphocytes, long-term survivors of treatment with F(ab')2 anti-CD4 and intact anti-CD4 were sacrificed for immunohistochemical analysis of their kidneys. Untreated B/W mice had large lymphocytic aggregates under the surface epithelium of the renal calyces. Most of these lymphocytes were CD4+ T cells, but CD8+ T cells and B cells were also present. In contrast, treatment with either intact anti-CD4 or F(ab')2 anti-CD4 substantially reduced, and in many cases prevented, the development of renal infiltrates. Treatment with either form of anti-CD4 not only inhibited renal infiltration by CD4+ T cells, but also prevented the accumulation of CD8+ T cells and B cells. These observations suggest a role for the CD4+ T cell in the accumulation of lymphocytes in target organs.

Intrathymic changes in murine CD3, CD5 and CD8 expression after in vivo administration of anti-CD4.

Intrathymic development of murine cortical and medullary thymocyte subpopulations was examined after in vivo administration of anti-CD4 mAb. Four days after mice received 1 mg anti-CD4, expression of CD4 was reduced to about 25% the levels of controls. On cortical CD8+/PNAhigh thymocytes, CD5 and CD8 expression both decreased, but not in parallel, whereas CD3 expression increased almost 2-fold. Partial shifts in CD3 expression were seen 3 and 6 h after injection, but modulation of CD4 preceded the increase in CD3 expression. On medullary CD8-/PNAlow thymocytes, both CD3 and CD5 were down regulated. On nontargeted CD8+/PNAlow medullary thymocytes, expression of these molecules was decreased less than 20%, but the decrease in CD8 expression was primarily due to the appearance of a CD8low subpopulation. The results indicate that anti-CD4 mAb differentially affects the intrathymic development of T cell populations.

A new model of Pneumocystis carinii infection in mice selectively depleted of helper T lymphocytes.

Pulmonary infections with Pneumocystis carinii are an important cause of morbidity and mortality in patients with AIDS. P. carinii infections are seen in patients with decreased numbers of helper T lymphocytes, suggesting that these cells are important in preventing infection. To test this hypothesis, we sought to establish experimental infection with P. carinii in mice selectively depleted of helper T lymphocytes. Weekly injections of a monoclonal anti-CD4 antibody produced sustained depletion of helper T lymphocytes from blood and lymphoid organs. To establish pulmonary infection, lymphocyte-depleted mice were then given intratracheal inoculations of P. carinii organisms derived from the lungs of chronically infected athymic mice. Pulmonary infection with P. carinii was demonstrable in the antibody-treated mice and was centered around the conducting airways. Infection was persistent for up to 3 mo with continued antibody treatments, and yet could be cleared from the lungs if antibody treatments were discontinued. This experimental model of P. carinii infection permits the study of infection associated with a specific immune defect and implicates the helper T lymphocyte as a critical cell in host defense against this pathogen.

Treatment of murine lupus with monoclonal antibody to L3T4. II. Effects on immunohistopathology of thymus, spleen, and lymph node.

Monoclonal antibody to L3T4 has been used successfully to suppress autoimmunity in the New Zealand black/New Zealand white F1 (B/W) mouse model for systemic lupus erythematosus. To clarify the immunopathology of murine lupus and determine the effects of anti-L3T4 treatment on the cellular composition and histopathology of lymphoid organs, we examined the distribution of lymphocyte subsets in cryostat sections of the thymus, spleen, and lymph nodes of B/W mice. Immunohistologic specimens were obtained from female B/W mice that had received weekly intraperitoneal injections of either rat monoclonal antibody to L3T4 (2 mg/mouse/week) or phosphate buffered saline (200 microliters/mouse/week) from age 5 months until euthanasia at 8 months. B and T cell domains in each organ were identified on serial sections with monoclonal antibody directed against B220 (all B cells), Thy-1.2 (all T cells), L3T4 (helper T cells), and Ly-2 (cytotoxic/suppressor T cells). In control mice, striking cytoarchitectural abnormalities were identified in the thymuses, and the spleen and lymph nodes were hypertrophied relative to anti-L3T4 treated mice. Thymic abnormalities included amplification of medulla, formation of thymomas, and cortical atrophy. Amplified medullary regions and thymomas in B/W mice contained numerous B cells and L3T4+ T cells but few Ly-2+ T cells. The enlarged spleens and lymph nodes of control mice consisted of numerous secondary follicles with germinal centers containing an unusual subpopulation of T cells that expressed L3T4 but not Thy-1.2. In contrast, mice treated with anti-L3T4 did not develop histopathologic changes characteristic of systemic lupus erythematosus in any organ. However, treatment depleted L3T4+ cells from the spleen and lymph nodes, and it modulated the expression of L3T4 by thymocytes. These observations demonstrate that treatment with anti-L3T4 not only interferes with L3T4-dependent T cell functions, but it also prevents progressive abnormalities in lymphoid tissue in lupus-prone B/W mice. This preservation of normal lymphoid structure may contribute to the beneficial effects of anti-L3T4 on autoimmunity.

Uptake and translocation of fluorescent latex particles by rabbit Peyer's patch follicle epithelium: a quantitative model for M cell uptake.

A quantitative, light microscopic morphometric model for uptake of particulates by Peyer's patch M cells was developed. Rabbit intestinal loops containing Peyer's patches were inoculated with fluorescent, non-degradable polystyrene microparticles (600-750 nm), and their localization in Peyer's patches was traced after varying time periods. The particles were localized sequentially at the FAE cell surface, spanning the entire width of FAE cells, and within the subepithelial dome as a function of time. The particles were associated with 5D9+ or 1D9+ M cells, but were not taken up or transported by villus epithelia. The kinetics suggested a synchronous wave of uptake and transepithelial transport. Quantitative analysis revealed a considerably greater uptake efficiency of polystyrene microspheres in comparison to other biological particles.