How Does Helicobacter pylori Cause Mucosal Damage? The Inflammatory Response

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How Does Helicobacter pylori Cause Mucosal Damage? The Inflammatory Response PETER B. ERNST,* SHEILA E. CROWE,T and VICTOR E. REYES*

* Departments of Pediatrics (Child Health Research Center), Microbiology and Immunology, and 'l'lnternal Medicine, University of Texas Medical Branch, Galveston, Texas

The role for Helicobacter pylori in the pathogenesis of disease provides the conundrum that only a subset of subjects infected with H. pylori will ever develop peptic ulcer or gastric cancer. Thus, variation in strain as well as environmental or host factors converge in the gastroduodenal milieu and control the final outcome of infection. The host immune and inflammatory response is emerging as an important element in the pathogenesis of these gastric diseases. The ideal host response provides protection to clear an infection without causing excessive amounts of inflammation that could compromise the integrity and function of host cells. This review will cover four main questions: (1) What are the mucosal immune/inflammatory responses that confer protection without damaging the host? (2) How do the gastric immune responses during infection with H. pylori differ from this ideal scenario? (3) Do these responses contribute to autoimmune-mediated damage to gastric tissue? (4) Can immunomodulation through vaccination enhance protective, nondestructive responses that prevent or treat infection or, at least, attenuate inflammation?

T

he ideal host response provides protection to clear an infection without causing excessive inflammation that could compromise the integrity and function of host cells. Current dogma seem to suggest that mucosal immune responses should select for secretory immunity that can cope with a luminal infection. These responses include secretory immunoglobulin (Ig) A. IgA synthesis and secretion is enhanced by regulatory T cells that produce a distinct pattern of cytokines. One model suggests that the pattern of cytokine production can distinguish two subsets of helper T cells, referred to as Thl and Th2 cells. Thl cells normally boost cellmediated immunity to cancers as well as intracellular infections, whereas Th2 cells seem to be more important in the generation of secretory immune responses in the mucosa. Paradoxically, during infection with H. pylori, an extracellular infection, Thl cells predominate, whereas Th2 cells are virtually absent. This would not be the ideal response for this type of infection and may therefore

account for the persistence of the infection. Moreover, Thl cells produce cytokines that promote inflammation, autoantibody formation, and cell-mediated damage to the epithelium. Direct evidence now exists to support the hypothesis that the immune/inflammatory responses observed during infection with H. pylori do, indeed, induce autoimmune-mediated damage to gastric epithelium. In contrast, oral vaccines can be designed so that Th2 cells are selected. These responses may not only diminish the bacterial load but also lead to the production of cytokines that attenuate inflammation and damage to gastric epithelial cells. Thus, the host response to natural infection, perhaps triggered by specific strains or regulated by specific host factors, can lead to an inappropriate immune response that fails to clear the organism and contribures to increased inflammation and damage to host tissue.

Goal of Mucosal Immune Responses The gastrointestinal immune system has the challenge of having to recognize and respond appropriately to pathogenic infections while not reacting excessively to dietary or microbial antigens that are habitually present in the lumen. The first step requires the host to recognize that it faces the potential for danger from a microbe that is a pathogen and not a commensal. We believe this process is triggered by "damage," a term that can be loosely defined as the ability of a pathogen to induce a nonspecific, acute inflammatory response. To fulfill the requirement that the host responds appropriately, regulatory T cells must be optimally activated. This model suggests that effective gastric immunity requires H. pylori to induce sufficient inflammation and damage to trigger a response. Based on the evident inflammatory response in the mucosa, there is little doubt that the organism has Abbreviations used in this paper: IFN, interferon; IL, interleukin; LFA, lymphocyte function-associated antigen; LPS, lipopolysaccharide; MHC, major histocompatibility complex; PigR, polymeric immunoglobulin receptor; TNF, tumor necrosis factor. © 1997 by the American Gastroenterological Association 0016-5085/97/$3.00

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caused the host to respond to the infection. The inability to clear the infection and the subsequent development of gastroduodenal disease probably results from inappropriate regulation by gastric T cells and their effects on immune and epithelial cell function.

Induction of Protective Immune Responses in the Gastrointestinal Tract Primary Inductive Sites As more and more is learned about the cellular and molecular basis for local immunity in the digestive tract, significant gaps in our knowledge are becoming apparent. Dogma suggests that the induction of immune responses in the intestine begins in the Peyer's patches. 1,2 Helper T cells in this inductive site produce cytokines that select for IgA production. Stimulated T and B lymphoblasts exit the Peyer's patches and seed other areas of the intestine as well as more remote mucosal tissues including the salivary glands and the respiratory and urogenital tracts. This model implies that lymphocytes from Peyer's patches would also seed the stomach; however, there is no direct evidence that this occurs. In fact, in the absence of gastric inflammation, very few T and B cells are found in the gastric mucosa, suggesting that the stimulation of intestinal Peyer's patches that occurs on a regular basis is insufficient to cause significant lymphocyte accumulation in the stomach. Recent observations suggest that solitary lymphoid nodules located at the base of the crypts throughout the mutine intestine may be a major source of intestinal T cells. 3 These so-called "cryptopatches" could produce T cells that seed other mucosal tissues, or, alternatively, similar structures may exist in other mucosal sites including the stomach.

Site-Specific Responses Cells within the Peyer's patches do not hold a monopoly on the ability to regulate immune and inflammatory responses in the digestive tract. This is shown by the fact that the uninfected human stomach has relatively few immune and inflammatory cells even though Peyer's patches are stimulated. However, in response to infection with H. pylori, there is a marked increase in the number of gastric lymphocytes. Factors within the effector sites, including antigen, chemokines, and adhesion molecules, are therefore required to enhance the accumulation and activation of immune and inflammatory cells that are associated with localized infections. Several investigators have shown that epithelial cells express cytokines as well as surface molecules that are

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necessary for white cell recruitment, adherence, differentiation, and activation. For example, after infection with invasive bacteria, intestinal epithelial cells produce interleukin (IL)-6, IL-8, granulocyte-macrophage colonystimulating factor, and monocyte chemoattractant protein 1(MCP-l).4 Stimulation of epithelial cells is associated with an increase in transcription of messenger RNA (mRNA) for IL-8 and subsequently results in a rapid increase in protein. s The epithelium can also respond to more subtle stress than that associated with invasion by bacteria or viruses. For example, bacteria or secreted products that bind to epithelium may be sufficient to activate this process. 6 ,7 The significant point is that epithelial cells transduce a signal to the host that reflects danger or damage and effectively focuses the host response to the site of infection and cellular stress.

Selection of Protective Responses Immunologic effector mechanisms may be driven by antigen, but the magnitude and type of immune response that develops is largely dictated by cytokines. Helper T cells can be divided into functional subsets based on their cytokine profile. Through the production of interferon gamma (IFN-)') and IL-2, Thl cells select for cell-mediated immunity, whereas Th2 cells are believed to preferentially regulate certain antibody responses, including mucosal IgA, through the production of IL-4, IL-5, and IL-6. 8 Because naive T cells retain the potential to differentiate into either subset, most antigens probably induce a mixture of T-cell responses. However, over time, certain pathogens may favor a response that mainly involves Thl or Th2 cells. Both subsets have been shown to playa pivotal role in immunity, and both have also been implicated in the immunopathogenesis of disease. 9 Thus, to maintain health, the host must strike the correct balance between T-cell subsets and/or the cytokine genes that are selectively expressed by these T cells. Antigen that is habitually present in the lumen often leads to oral tolerance that is mediated by regulatory T cells in the Peyer's patches, including Th2 cells.lO It is not clear what controls the selection of immunity vs. tolerance, but factors such as route of antigen administration, dose, site of antigen presentation, fluxes in antigen load, and degree of inflammation at the inductive or effector site may all affect the eventual outcome. Th2 cells seem to be a desirable regulatory cell in the mucosa because they satisfy the criterion of selecting for protective mucosal IgA responses while inhibiting cellmediated immunity. The chronic mucosal inflammation in association with a persistent exposure to H. pylori intimates that Thl cells predominate in the inflamed

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stomach and subvert the strategy of developing protection without inflammation.

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Immunologic Events in the Stomach During Infection With H. pylori It is well known that in adults, H. pylori induces a chronic-active inflammation with a mixture of neutrophils, T cells as well as B cells, and plasma cells. These cellular changes are associated with an increase in IL-1, IL-6, IL-8, and tumor necrosis factor (TNF)-a concentrations l l - 13 and, more recently, in the number of IFN-,),producing cells. 14 Although these responses apparently do little to confer protective immunity against H. pylori, they can induce a number of changes in the gastric epithelium that may promote inflammation and epithelial cell damage. In turn, damaged epithelial cells may increase the risk of peptic ulcer or lead to aberrant repair recognized as gastric atrophy or epithelial cell metaplasia. For example, cytokines, including TNF-a, IFN-')', and IL-1, can increase the expression of molecules by the gastric epithelium including lysozyme, IL-8, class II major histocompatibility complex (MHC) molecules, and the polymeric immunoglobulin receptor (PIgR) (reviewed by Brandtzaeg et al.,IS Ernst et al.,16 and Dixon 17). Interestingly, specific alleles of class II MHC molecules have been suggested to playa role in determining risk for gastroduodenal disease. 18

Activation of the Acute Inflammatory Response Gastritis can occur in the absence of T and B cells after infection of immunodeficient mice with Helicobacter felis. 19 This suggests that at least some of the changes in patients infected with H. pylori result from nonspecific inflammatory responses. It is possible that neutrophil activation is proportional to the load of H. pylori and results in damage to the epithelium either through the production of oxidants 20 ,21 or as a consequence of transepithelial migration 22 (Figure 1). In addition, neutrophil adherence to the endothelium in gastric tissue could lead to protein leakage as a result of microvascular damage. 23 Although H. pylori is rarely invasive, proinflammatory material may be shed and reach the underlying fibroblasts and inflammatory cells, particularly if the epithelial barrier function is compromised. The gene coding for a novel neutrophil-activating protein has recently been cloned from H. pylori and shown to enhance the binding of neutrophils to endothelium. 24 Formylated tripeptides, lipopolysaccharide (LPS),2S urease,26 or other factors produced by the bacterium may also lead to changes in the expression of surface adhesion molecules 27 as well as

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Figure 1. An overview of immune-mediated damage in gastric disease. Several immune and inflammatory reactions probably contribute to damage in gastric and duodenal tissue. First, the infection and associated cytokines induce chemokines such as IL-8 and growth-regulated gene a(Gro-a) that, in turn, lead to neutrophil accumulation and activation. Subsequent transepithelial migration and release of neutrophil mediators can disrupt epithelial cell function. Second, gastric B cells detected during infection with H. pylori are often autoreactive. Moreover, many of these antibodies are IgG and capable of activating complement. Local antigenantibody complexes and activated complement (C') would be highly destructive to the epithelium and underlying tissue. Finally, gastriC T cells, discussed in detail in Figure 2, become activated by the epithelial cells and could contribute to the local damage byenhancing B-cell growth as well as directly damaging gastriC epithelial cells. Although these pathways have been well studied in the stomach, it remains to be determined if they are relevant in the duodenum in association with duodenal ulcer. PMN, polymorphonuclear leukocyte; ENA-78, epithelial cell-derived neutrophilactivating peptide 78.

the migration,26 adhesion,24 and activation of neutrophils 28 ,29 or monocytes. 30 ,31 Stimulated monocytes/ macrophages and fibroblasts produce IL-1, IL-6, or TNF-a that would also contribute to the cytokine milieu during H. pylori infection. 32 Crabtree et al,33,34 have conducted several studies documenting the ability of H. pylori to activate neutrophils by stimulating IL-8 production by the gastric epithelium. These original observations have been extended in other reports showing that H. pylori induces the accumulation of mRNA for IL-8 and stimulates the secretion of immunoreactive as well as biologically active IL-8 from gastric epithelial cell lines. 3s - 37 This in vitro evaluation correlates with the observed increase in neutrophils and IL-8 in the mucosa. 26 ,38 Moreover, H. pylori induces IL-8 without invasion in gastric cell lines,3s suggesting that the natural tropism between H. pylori and gastric epithelium is mimicked in these in vitro approaches. The mechanism of the epithelial response to H. pylori has yet to be established. Experiments by Keates et al. 39 suggest that H. pylori activates the transcription factor NF-KB, which in turn could be responsible for important

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intracellular signaling events in the production ofIL-8. 39 The role for live bacteria in the induction of IL-8 production by gastric epithelial cells has been suggested by several reports because killed H. pylori did not stimulate IL-8 production. 35 ,36 However, another report indicates that secreted products from H. pylori do stimulate IL-8 production, particularly in nongastric cellsY This apparent discrepancy may result from the use of different strains of H. pylori and the length of time used for cell stimulation. The effect of bacterial strain on the IL-8 response became apparent when H. pylori bearing the cagA gene were associated with more severe gastric pathology and IL-8 production. This conclusion was based on the fact that natural mutants lacking cagA did not induce IL-8 in vitro. 4o Another study shows that isogenic murants lacking cagA and/or vacA are still capable of inducing IL_8. 36 More recently, the ability of H. pylori to control IL-8induction has been ascribed to genes in proximity to cagA in a region referred to as the pathogenicity island. For example, disruption of the picB gene has been shown to abrogate the induction of IL-8. 41 Thus, genes associated with the induction of inflammation may also be linked to or directly responsible for the production of virulence factors that will affect the outcome of infection with H. pylori.

Activation of T and B Cells H. pylori has been shown to be a modest T-cell mitogen in vitro and, in fact, may even reduce some T-cell responses. 42 It is possible that one of several molecules expressed by H. pylori, including proteases, may interfere with antigen presentation and T-cell activation. However, the striking accumulation ofT cells in the gastric mucosa during infection with H. pylori seems to support the fact that T cells are recruited and activated in vivo. Although it is not clear what proportion of the infiltrating T cells are specific for H. pylori, MHCrestricted, H. pylori-specific T-cell clones have been derived from both the peripheral blood and gastric mucosa of infected individuals. 43 It is not clear if gastric T cells arise as a result of local expansion or recruitment from other inductive sites such as the Peyer's patches. However, recent evidence suggests that the gastric epithelium has an unusual propensity to stimulate helper T cells. In addition to expression of class II MHC molecules during infection with H. pylori, gastric epithelial cells also express accessory molecules that facilitate T-cell activation. For example, gastric epithelial cell lines as well as epithelial cells freshly isolated from gastric biopsy specimens of infected donors, express lymphocyte function-associated antigen (LFA)-3 (Haeberle et al., manuscript submitted) and B7.2.44 Both of

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these molecules are increased during infection and are important for the stimulation of T cells by antigenpresenting cells. In this case, gastric epithelial cells seem to be competent antigen-presenting cells because they are capable of internalizing and processing antigen as well as stimulating CD4+ T cells. These molecules are not only associated with T-cell activation, but also modulate T-cell function. For example, LFA-3 seems to help activate Thl cells and cell-mediated immunity because blocking its interaction with its counterreceptor on T cells (CD 2) leads to an inhibition of cell-mediated immune responses. 45 Thus, the increase in LFA-3 on gastric epithelial cells during infection with H. pylori may help select for Thl cells. In support of the notion that Thl cells are produced during infection are data showing that H. pylori induces IFN -"{ production by mononuclear cells isolated from peripheral blood 46 or gastric mucosa. 42 Furthermore, IFN-"{-producing cells are increased in the gastric mucosa during infection. 14 This latter study also suggested that Thl cells predominate in gastritis because the number of IL-4-producing Th2 cells varied little in response to inflammation. 14 Recently, Bamford et al. showed that both gastric CD4+ and CD8+ T cells produce IFN-"{ and IL-2 and not IL-4 or IL-5, thereby confirming the enrichment of Thl-like cells during infection (Bamford et al., manuscript submitted). It should be noted that the increase in IFN-"{-producing cells was also observed in gastritis not caused by H. pylori. 14 It is not clear if Thl responses, which select for cell-mediated immunity, are in the best interest of the host in its attempt to clear an extracellular infection. Moreover, Th2 responses have been directly implicated in mucosal protection as well as split tolerance, i.e., enhanced IgA responses in association with suppressed cell-mediated immunity. Thl cells and the associated cell-mediated immunity may be tolerable in controlled amounts, but an overwhelming Thl response, in combination with host genetics, specific strains of bacteria, as well as other environmental factors, could combine to contribute to epithelial damage and eventually ulcerogenesis. These interactions may explain why different clinical manifestations are observed in a family infected with the same strain of H. pylori.

AutOimmunity and H. pylori B Cells and Induction of Gastritis The first suggestion that H. pylori may cause a bona fide autoimmune response was based on evidence that monoclonal antibodies directed against H. pylori

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could recognize an epitope on the gastric epithelium of mice and humans. Moreover, administration of these antibodies to mice resulted in gastritis and caused mild erosions. 47 Since that report, IgM antibodies produced from immortalized B cells obtained from the gastric mucosa have been shown to recognize the gastric epithelium. 48 More recently, an elegant series of experiments has shown that H. pylori lPS expresses lewis antigens, and cross-reactive antigens are expressed by epithelial cells. 49 Antibodies to H. pylori or its lPS can recognize gastric epithelial cells. Moreover, antibodies to lewis antigens can induce gastritis when administered to mice. 49 Other evidence suggests that B cells within a MAlToma express a repertoire that recognizes a determinant shared by both IgA and IgM.50 Thus, antibodies within the gastric mucosa that arise in response to infection recognize epithelial cells or act as rheumatoid factors. This could lead to immune complex-mediated disease that directly damages the epithelium (Figure 1). Indeed, activated complement has been shown adjacent to gastric epithelium in gastritis. 51

T Cell-Induced Epithelial Cell Damage Through T-cell regulation, most luminal flora stimulate a balance among tolerance, inflammation, and immunity, thereby limiting tissue damage. The failure to accomplish this during infection with H. pylori suggests that an imbalance in Th cell cytokines may shift the host response away from protective immunity and more towards chronic inflammation and tissue damage. The evidence that Th 1 cells may be increased relative to Th2 cells during H. pylori infection suggests that a marked skewing in this response may lead to disease as implicated in the pathogenesis of more classic autoimmune diseases. 9 The presence of Thl cells and IFN--y production are likely to lead to immunophysiological interactions that directly promote tissue damage. For example, IFN--y alters epithelial barrier function in intestinal cell lines. 52 Other cytokines, including TNF-a, can collaborate with IFN--y to alter epithelial cell Il-8 gene expression. 53 In addition, Thl cells enhance cell-mediated immune responses that could directly contribute to epithelial cell death at levels that exceed normal tissue repair mechanisms (Figure 2). Cell death can occur by necrosis or apoptosis. In contrast to necrosis, apoptosis is a programmed form of cell death in which DNA fragments resulting from cleavage at the internucleosomal junctions are generated. These fragments are in multiples of 180 base pairs and can be detected in several assays of apoptosis. Apoptosis is detected at low levels in normal intestinal epithelium but is increased in melanosis coli, graft-versus-host disease,

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Immune Activation By Epithelium

Ahered Epithelial

Function

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Epithelial Death. Atrophy, and Metaplasia

Figure 2. T cell-mediated damage of gastric and duodenal epithelium. This figure illustrates the role of T cells as one of the many immune and inflammatory mechanisms involved in the pathogenesis of gastroduodenal disease. Initially, the epithelial barrier is sufficiently tight so that luminal acid or enzymes are of little consequence (left). However, over time, inflammation progresses and a significant accumulation of Th1 cells occurs. This leads to the production of cytokines such as IFN-'Y and IL-2 that promote cell-mediated immunity. Through these cytokines, as well as Fas-Fas ligand interactions, the host response combines with the bacteria to increase cell-mediated immunity (middle) and epithelial cell death via apoptosis (right). The consequences of increased cell death include aberrant repair of the epithelium, which may lead to atrophy and/or metaplaSia. This abnormal tissue would lack the full complement of cytoprotective mechanisms and may increase the risk of peptiC ulceration.

nonsteroidal anti-inflammatory drug-induced enteropathy, and human immunodeficiency virus infection and with chemotherapy or radiation. 54 Defective apoptosis is thought to play a role in carcinogenesis because certain genes that are associated with the development of colorectal cancer (c-myc,p53 , and bcl-2) regulate apoptosis. Apoptosis has not been as well studied in the stomach, but several reports suggest that H. pylori infection is associated with increased apoptosis. Recently, Moss et al,55 reported that apoptotic cells were rare in uninfected gastric tissue samples with a mean of 2.9% of epithelial cells located in the most superficial aspect of the gastric glands. In infected tissues, apoptotic cells were located throughout the depth of the gastric glands and increased in mean numbers (16.8%), a value that decreased to 3.1 % after H. pylori eradication. A preliminary study by Jones et al. 56 has confirmed increased epithelial apoptosis in gastric biopsy specimens from H. pylori-infected patients, which also decreased after successful eradication therapy. The frequency of epithelial apoptosis was found to be significantly lower in noninflamed mucosa in this study. Another initial report also describes increased apoptosis of epithelial cells in the neck region of the gastric glands of H. pylori-infected subjects that decreased after eradication therapy. 57 Immunohistochemical staining for inducible nitric oxide synthase expression and for a marker of

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peroxynitrite formation was also increased before H. pylori treatment in this study. Although these three studies support the concept that H. pylori infection is associated with increased epithelial apoptosis, Peek et aps suggest that this effect may be due to cagA-negative strains because their data show increased apoptosis in biopsy specimens from patients infected with cagA-negative strains. In contrast, cagA-positive strains were associated with increased epithelial proliferation, but apoptosis was not different from uninfected controls. Most of the subjects reported as having an increase in apoptosis in the other studies 55 - 57 were probably infected with cagApositive strains however. Thus this issue remains to be clarified. Cell-mediated immune responses are well known to induce apoptosis of target cells, leading to the prediction that the heightened gastric Th1 response would contribute to epithelial damage. Recent results suggest that cytokines from Th1 cells enhance cell death in gastric epithelial cells through apoptosis. Additional intercellular interactions between epithelial cells and T cells mediated by Fas-Fas ligand interactions may also contribute to apoptosis of epithelial cells. This process may be important in regulating cell death and preventing cancer, but it could also induce breaks in the epithelial barrier that cause erosions, which may eventually lead to an increased risk of developing peptic ulcers. From another perspective, the excessive rate of epithelial cell turnover associated with the increase in apoptosis may eclipse the capacity of tissue repair mechanisms to properly replace damaged epithelial cells. In turn, epithelial atrophy and metaplasia may ensue. Additional research into these various possibilities remains to be performed.

Strategies in Immunotherapy We propose that an excessive Th1 response driven by a natural infection with H. pylori would favor IFN-I' production, the development of cell-mediated immunity, and a set of conditions that favor the onset of epithelial damage. This damage may be direct, through the recognition of immunogenic peptides presented by the epithelial cells, or by the transient or permanent expression of epitopes that are recognized by antibodies produced during infection. Epithelial damage may also occur indirectly. For example, TNF-a, IL-1, IFN -I', and other cytokines may augment the expression of IL-8 or adhesion molecules that enhance neutrophil rectuitment, activation, and transepithelial migration. Thus, the epithelium may be damaged directly by cell-mediated immune mechanisms or as bystanders inadvertently exposed to cytotoxic material from activated neutrophils

or macrophages that are attempting to phagocytose bacteria or bacterial products. If this model is correct, then immunologic intervention that shifts the T-cell response from Th 1 to Th2 may favor the development of IgA responses and protective immunity in association with a decrease in tissue damage. This notion is supported because IgA antibodies recognizing urease are sufficient to provide protection. 59 In addition, in contrast to natural infection, protective immunity can be achieved by immunizing mice with H. pylori antigens, in combination with an adjuvant such as cholera toxin. This approach provides protection against a challenge with Helicobacter felis or H. pylori60- 62 (for review, see Ghiara and Michetti 63 ). These data further support the hypothesis because cholera toxin boosts Th2 cells and IgA responses. s Thus, qualitatively changing the response through oral immunization with the correct antigenic subunits and adjuvants provides a tremendous opportunity for prevention as well as a complement to therapy. Future experiments will have to determine the relative strength of the Th 1 and Th2 cell responses in different stages of gastric disease vs. the response induced by the numerous candidate oral vaccines that are being developed.

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55. Moss SF, Calam J, Agarwal B, Wang S, Holt PG. Induction of gastric epithelial apoptosis by Helicobacter pylori. Gut 1996;38: 498-501. 56. Jones NL, Yeger H, Cutz E, Sherman PM. Helicobacter pylori induces apoptosis of gastric antral epithelial cells in vivo (abstr). Gastroenterology 1996;110(Suppl):A933. 57. Mannick EE, Bravo LE, Zarama G, Realpe JL, Zhang X-J, Ruiz B, Fontham ETH, Mera R, Miller MJS, Correa P. Inducible nitric oxide synthase, nitrotyrosine and apoptosis in Helicobacter pylori gastritis: effect of antibiotics and antioxidants. Cancer Research 1996;56:3238-3243. 58. Peek RM, Moss SF, Tham KT, Perez-Perez GI, Miller GG, Holt PR, Blaser MJ. Infection with H. pylori cagA+ strains dissociates gastric epithelial proliferation from apoptosis (abstr). Gastroenterology 1996;110(Suppl):A575. 59. Blanchard TG, Czinn SJ, Maurer R, Thomas WD, Soman G, Nedrud JG. Urease-specific monoclonal antibodies prevent Helicobacter felis infection in mice. Infect Immun 1995;63:13941399. 60. Chen M, Lee A, Hazell S. Immunisation against gastric Helicobacter infection in a mouse/Helicobacter felis model. Lancet 1992;339:1120-1121. 61. Michetti P, Corthesy-Theulaz I, Davin C, Haas R, Yaney A-C, Heitz M, Bille J, Kraehenbuhl J-P, Saraga E, Blum AL. Immunization of BALB/c mice against Helicobacter felis infection with Helicobacter pylori urease. Gastroenterology 1994;107:10021011. 62. Pappo J, Thomas WD, Kabok Z, Taylor NS, Murphy JC, Fox JG. Effect of oral immunization with recombinant urease on murine Helicobacter felis gastritiS. Infect Immun 1995;63:1246-1252. 63. Ghiara P, Michetti P. Development of a vaccine. Curr Opin GastroenteroI1995;11:52-56. Received June 30, 1997. Accepted July 22, 1997. Address requests for reprints to: Peter B. Ernst, D.V.M., Ph.D., Department of PediatriCS, Children's Hospital, Route 0366, 301 University Boulevard, Galveston, Texas 77555-0366. Fax: (409) 772-1761.