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Helicobacter pylori and gastric autoimmunity
Microbes and Infection 6 (2004) 1395–1401 www.elsevier.com/locate/micinf
Review
Helicobacter pylori and gastric autoimmunity Mario Milco D’Elios a,*, Mathijs P. Bergman b,c, Amedeo Amedei a, Ben J. Appelmelk b, Gianfranco Del Prete a a Department of Internal Medicine, University of Florence, Viale Morgagni, 85-50134 Florence, Italy Department of Medical Microbiology, Vrije Universiteit, Medical School, van der Boechorstraat 7, 1081 BT Amsterdam, The Netherlands c Department of Medical Microbiology and Infectious Diseases, Erasmus Medical Center, Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands b
Available online 05 November 2004
Abstract Host specific T-cell response is critical for the outcome of Helicobacter pylori infection. In genetically susceptible individuals, H. pylori can activate gastric CD4+ Th1 cells that recognize cross-reactive epitopes shared by H. pylori proteins and self H+, K+-ATPase, leading to gastric autoimmunity via molecular mimicry. © 2004 Elsevier SAS. All rights reserved. Keywords: Autoimmunity; Mimicry; Mucosal immunity; Th1–Th2 response; Cytokines; Helicobacter pylori; Atrophic gastritis; Apoptosis; H+, K+-ATPase
1. Introduction Helicobacter pylori is a Gram-negative bacterium that infects half the world’s population. H. pylori infection may remain asymptomatic lifelong or result in different clinical outcomes, such as chronic active gastritis, peptic ulcer, mucosal atrophy, gastric cancer, or gastric B-cell lymphoma of the mucosa-associated lymphoid tissue (MALT) [1]. Autoimmune—type A—gastritis (AIG) is an organspecific inflammatory disease, which usually does not result in overt symptoms until development of gastric atrophy or pernicious anemia (PA). AIG gastritis, which involves the gastric corpus and fundus, but not the antrum, is characterized by (a) serum autoantibodies to gastric H+, K+-ATPase, (b) high serum gastrin levels, (c) decreased acid secretion, (d) decreased pepsinogen I/II ratio and (e) frequent association with thyroid or other endocrine autoimmune disorders [2]. Gastric atrophy, characterized by loss of corpus and antrum glands, is considered a precursor of gastric adenocarcinoma. Since H. pylori increases the risk of gastric adenocarcinoma, it has been classified as a class I oncogenic factor [3]. The mechanisms involved in the development of mucosal atrophy are still unknown. H. pylori-related atrophic gastritis and * Corresponding author. Tel.: +39-05-5429-6445; fax: +39-05-5427-1494. E-mail address: [email protected] (M.M. D’Elios). 1286-4579/$ - see front matter © 2004 Elsevier SAS. All rights reserved. doi:10.1016/j.micinf.2004.10.001
AIG, which share a number of clinical, epidemiological and immunological characteristics, have recently been the matter of investigation [4–6]. This article will focus on the experimental evidence suggesting that H. pylori infection is involved in the genesis of gastric autoimmunity via molecular mimicry and the activation of T cells that mediate gastric damage.
2. H. pylori infection, gastric autoantibodies and atrophic gastritis Half of H. pylori-infected individuals (range 49–64%) have serum autoantibodies reactive to gastric parietal cells [7,8]. H. pylori colonization takes place in the antrum, while infected humans and experimentally infected mice develop atrophy in the corpus. The mechanisms involved in the genesis of H. pylori-associated atrophic gastritis are still poorly known. In humans, a series of observations suggests that gastric corpus atrophy can be related to an autoimmune process driven by H. pylori. Immunohistochemistry analysis of human normal gastric tissue incubated with serum from H. pylori-infected patients revealed that two different sites of gastric mucosa are targets of autoantibodies: one on the luminal membranes of the foveolar epithelial cells in the antrum and corpus mucosa, and the other one on the canalicular membranes of parietal cells in the gastric corpus mu-
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cosa [8]. Parietal cells secrete gastric acid, and their apical secretory canaliculi are very rich in H+, K+-ATPase, the gastric proton pump. The gastric H+, K+-ATPase has been identified as the major target autoantigen in chronic H. pylori gastritis with corpus atrophy [9]. Sera of H. pylori-infected mice cross-react with the human gastric mucosa, and these cross-reactive antibodies can be removed by pre-incubation with H. pylori cells because lipopolysaccharide (LPS) of H. pylori expresses Lewis blood group (Le) antigens similar to those expressed on human cells, including gastric epithelial cells [10]. In mice, H. pylori infection can induce autoantibodies through mimicry of Lewis antigens on the gastric proton pump [11]. H. pyloriinfected mice develop antibodies to H. pylori LPS, to Lewis x (Lex) and Ley and to murine H+, K+-ATPase. In mice, Lex and Ley are expressed on gastric mucin, whereas Ley is expressed on the b-subunit of gastric H+, K+-ATPase. In humans, however, serum autoantibodies recognize both the native and the recombinant H+, K+-ATPase lacking Lewis antigens [9], and they cannot be removed by pre-incubation with H. pylori cells [11]. Therefore, human anti-H+, K+ATPase autoantibodies associated with H. pylori infection do not involve Lewis mimicry, as it is for Helicobacter infection in mice [12]. Both antiluminal and anticanalicular antibodies are significantly associated with H. pylori infection. However, in H. pylori-induced atrophic gastritis, only anticanalicular autoantibodies positively correlate with clinical parameters and severity of H. pylori gastritis in the corpus, as well as with atrophy and increased corpus apoptosis [8,13]. The a- and b-subunits of H+, K+-ATPase are the targets of anticanalicular autoantibodies, and patients with H+, K+ATPase autoreactivity show more severe mucosal atrophy [9]. In addition, H. pylori-infected patients with anticanalicular autoantibodies have significantly higher serum gastrin levels and lower pepsinogen I:II ratio, which are considered markers for corpus atrophy [8,14]. The presence of anticanalicular autoantibodies correlates with decreased gastric acid output in non-ulcer dyspepsia patients infected with H. pylori [14]. On the basis of the similar histopathological and clinical characteristics of H. pylori-associated atrophic gastritis with anticanalicular antibodies and classical AIG, it has been suggested that H. pylori can represent a causative agent of gastric autoimmunity [11]. In support of this hypothesis are the observations that after H. pylori eradication, gastric autoantibodies decrease, and histologically defined AIG can be successfully reversed in most infected patients [15,16]. However, individuals with severe long-lasting atrophic corpus gastritis do not have amelioration of atrophy or intestinal metaplasia after cure of H. pylori infection, though achieving increased gastric acid secretion and reduced hypergastrinemia [17]. A strong association between H. pylori infection and gastric autoimmunity is supported by studies indicating that most patients with AIG have or have had H. pylori infection. Actual H. pylori colonization could be detected by histology
in only 10–14% of PA patients and 33% of individuals with atrophic corpus gastritis, and a single study reported even a negative association between H. pylori and PA. The conflicting prevalence of H. pylori infection in AIG/PA can be largely ascribed to the methods (histology/breath test vs. serology) used for defining H. pylori infection. Serum antibodies to H. pylori were measured in 51–83% of patients with PA and in 53–86% of patients with atrophic corpus gastritis, providing indirect support to the concept that a remarkable proportion of patients with atrophic gastritis have had H. pylori gastritis before the pathogen was cleared by the development of hypochlorydria and atrophy (reviewed in [18]).
3. T-cell responses in H. pylori-infected patients with AIG H. pylori-associated atrophic gastritis is characterized by an inflammatory infiltrate in the gastric mucosa, including T cells, macrophages and B cells. It mainly affects the corpus and the fundus, like classical AIG, and time after time it is accompanied by loss of gastric parietal and zymogenic cells. In the last few years, we characterized at the molecular level the gastric T-cell-mediated responses to H. pylori and to the H+, K+-ATPase autoantigen in a series of H. pylori-infected patients with gastric autoimmunity [6]. The clonal progenies of in vivo activated gastric T cells recovered from gastric biopsies were screened for their ability to proliferate in response to H. pylori and to H+, K+ATPase. Among gastric CD4+ helper T-cell (Th) clones, a number proliferated to H. pylori, but not to the recombinant H. pylori proteins CagA, VacA, hsp, urease nor to H+, K+ATPase. Some other Th clones proliferated in response to H+, K+-ATPase and not to H. pylori (autoreactive), and a third group of clones was found that proliferated to both H. pylori and H+, K+-ATPase [6] (Table 1). All the Th clones able to proliferate to H+, K+-ATPase were studied for their ability to respond to 205 overlapping 15-mer peptides covering the amino acid sequence of the a chain and 56 peptides for the b chain of the human H+, K+-ATPase. In the series of 13 clones that proliferated to both H+, K+-ATPase and H. pylori lysate, 11 recognized their epitope in the a chain, and two clones in the b chain. In the subgroup of Th clones that proliferate exclusively to H+, K+-ATPase, six recognized their epitope in the a chain and nine in the b chain of the proton pump. It is of note that a substantial proportion of those epitopes of H+, K+-ATPase are almost the same as those that are the target of autoreactive gastric T-cell response in AIG patients without evidence of concomitant H. pylori infection [4,5]. No overlap indeed was found between the H+, K+-ATPase epitopes recognized by clones reactive to H+, K+-ATPase only and the H+, K+-ATPase epitopes recognized by clones able to proliferate to both H+, K+-ATPase and H. pylori lysate (Table 1) [6]. Therefore, a number of H+, K+-ATPase epitopes can be considered as “private”, whereas other (“shared”) epitopes,
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Table 1 Differences in H+, K+-ATPase epitope specificities between cross-reactive and autoreactive gastric Th clones from H. pylori-infected AIG patients Th clones reactive to both H+, K+-ATPase and H. pylori (cross-reactive) H+, K+-ATPase ␣ chain peptides recognized PA.P24 (Vb4) a46–60 FF.A15 (Vb18) a181–195 FF.C32 (Vb16) a241–255 FF.C27 (Vb5.2) a256–270 FF.C26 (Vb21.3) a516–530 GR.A12 (Vb19) a576–590 GR.C31 (Vb13) a621–635 FF.A05 (Vb16) a621–635 GR.A04 (Vb13) a781–795 PA.R37 (Vb15) a836–850 MI.A30 (Vb13) a836–850 H+, K+-ATPase b chain peptides recognized FF.C13 (Vb14) b11–25 GR.C27 (Vb13) b216–230
T-cell clones reactive to H+, K+-ATPase (autoreactive) PA.Q08 (Vb4) GR.C11 (Vb5.3) PA.P34 (Vb19) FF.C39 (Vb9) PA.P02 (Vb8) PA.P14 (Vb19)
a1–15 a31–45 a151–165 a351–365 a881–895 a881–895
FF.A09 (Vb23) FF.A33 (Vb6.7) FF.C15 (Vb22) GR.A01 (Vb6.7) FF.C03 (Vb16) PA.R17 (Vb4) GR.C26 (Vb13) MI.A42 (Vb8) MI.B46 (Vb17)
b76–90 b76–90 b76–90 b81–95 b81–95 b111–125 b166–180 b231–245 b231–245
H+, K+-ATPase epitopes recognized by gastric Th clones derived from AIG patients with current H. pylori infection. Cross-reactive Th clones recognize some H+, K+-ATPase epitopes, whereas autoreactive Th clones recognized other H+, K+-ATPase epitopes. The gastric clones are indicated by the acronime of the donors (GR, PA, MI and FF), and the respective TCR Vß chain expression is reported.
Table 2 H. pylori and H+, K+-ATPase peptides recognized by gastric cross-reactive Th clones Th clone
GR.C31 GR.A04 PA.P24 PA.R37 MI.A30 FF.A15 FF.C32 FF.C27 FF.C26 FF.A05
Amino acid sequences recognized H. pylori cross-reactive peptide H+, K+-ATPase epitope VRVDVRRLDHLMNLI IRVIMVTGDHPITAK ISNLPYYIATRLVLN NLKKSIAYTLTKNIP LNNYQKENSLYNHNL KKEMEINDHQLSVAE NMRVFIIHLSPKTCK KAESDIMHLRPRNPK NMRVFIIHLSPKTCK KAESDIMHLRPRNPK VVQGGDKFHAPVLVD VIRDGDKFQINADQL VIQIGPMPTPAIAFL CTHESPLETRNIAFF ALDSLEKVVARLVVK STMCLEGTAQGLVVN VFKGIPGLSLEAVEK VMKGAPERVLERCSS IRIVKTTGDKILDAP IRVIMVTGDHPITAK
Amino acid sequence position H. pylori protein autoantigen 264–278 histidine kinase 621–635 a H+, K+-ATPase 99–113 dimethyl adenosine transferase 781–795 a H+, K+-ATPase 104–118 penicillin-binding protein 2 46–60 a H+, K+-ATPase 11–25 LPS biosynthesis protein 836–850 a H+, K+-ATPase 11–25 LPS biosynthesis protein 836–850 a H+, K+-ATPase 93–107 acetate kinase 181–195 a H+, K+-ATPase 70–84 phosphoglucosamine mutase 241–255 a H+, K+-ATPase 78–92 virb4 homolog 256–270 a H+, K+-ATPase 571–585 glucose-inhibited division pA 516–530 a H+, K+-ATPase 35–49 porphobilinogen deaminase 621–635 a H+, K+-ATPase
For each gastric Th clone reactive to both H. pylori and H+, K+-ATPase, one H. pylori cross-reactive peptide and one H+, K+-ATPase epitope were identified. Identical amino acid residues in the cross-reactive H. pylori and H+, K+-ATPase peptides recognized are highlighted.
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Fig. 1. Human H+, K+-ATPase and H. pylori mimic T-cell epitopes identified in gastric autoimmunity.
mainly in the a chain, are cross-reactive with epitopes of H. pylori antigens.
4. T-cell-mediated mucosal damage in gastric autoimmunity
Of the cross-reactive Th clones, two recognized the a836– 850 epitope, and two other ones from different patients recognized the a621–635 epitope (Table 2). A cross-reactive H. pylori peptide could be identified for 10 H+, K+-ATPase/H. pylori cross-reactive gastric Th clones [6]. Two clones from different patients, which shared recognition of the a836– 850 H+, K+ATPase epitope, also shared cross-reactivity to the 11–25 peptide of a LPS biosynthesis protein of H. pylori (Table 2). Of two other clones from different patients, both reactive to H+, K+-ATPase a621–635, one proliferated to the H. pylori histidine kinase 264–278 peptide, whereas the other showed cross-recognition of the H. pylori porphobilinogen deaminase 35–49 epitope. Overall, that study led to the identification of nine H. pylori proteins, each harboring a T-cell peptide suitable for cross-reaction with T-cell epitopes of gastric H+, K+-ATPase a chain (Fig. 1). Interestingly, none of the bacterial epitopes recognized by cross-reactive T-cell clones belong to the known H. pylori immunodominant antigens, such as CagA, VacA and urease, which are major targets of gastric T-cell responses in H. pylori-infected patients with peptic ulcer [19]. The sequence homology between cross-reactive H. pylori and H+, K+-ATPase peptides varies and can be more or less stringent (Table 2), considering that degeneracy in both TCR and MHC binding motifs can reduce sequence-specific requirement to only a few crucial residues [20]. A clear example is provided by the animal model of myocarditis, where cross-recognition of the autoantigen (e.g. myosin) and a chlamydial peptide depends only on four identical residues in the amino acid sequence [21].
Upon stimulation with the relevant peptide, all crossreactive Th clones and most of the H+, K+-ATPase-specific Th clones from the gastric corpus of H. pylori-infected AIG patients produced IFN-c, but not IL-4 (Th1 cytokine profile) [6]. All the cross-reactive and autoreactive H+, K+-ATPasespecific clones also produced high concentrations of TNF-a. Production of IFN-c, and TNF-a by Th1 cells can locally act, increasing the expression by gastric epithelial cells of both MHC class II molecules and B7-1 (CD80) or B7-2 (CD86) costimulatory molecules, as well as cathepsins involved in antigen processing, thus favoring the presentation of peptides by such non-professional antigen-presenting cells (APCs) [18]. Following activation, virtually all gastric cross-reactive and autoreactive Th clones were able to induce cell death via either Fas–Fas ligand-mediated apoptosis or perforinmediated cytotoxicity against target cells which they were in close contact with [4,6]. Parietal cells can express Fas (CD95) molecule, and HLA-DR is ectopically expressed on glandular epithelium in proximity to T-cell infiltrates in human AIG [12,18]. Requirement for Fas in development of full-blown experimental autoimmune gastritis (EAIG) has been demonstrated in the model of lpr/lpr mice, which are deficient in Fas expression and fail to develop destructive gastritis and autoantibody production upon neonatal thymectomy [22]. Up-regulated Fas expression by gastric parietal cells in EAIG mice may explain, at least in part, the selective destruction of parietal cells [22]. Based on these observations, it is tempting to speculate that in the inflammatory scenario in which cross-reactive and autoreactive Th clones
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5. What gives the start to gastric autoimmunity?
Fig. 2. Model of T-cell-mediated damage in H. pylori infection with gastric autoimmunity. H. pylori induces in susceptible individuals the expansion of H. pylori-specific Th1 cells that cross-react with H+, K+-ATPase epitopes. H. pylori-H+, K+-ATPase cross-reactive Th1 cells could mediate destruction of gastric mucosa, via Fas-ligand (Fas-L)-induced apoptosis and perforinmediated cytotoxicity.
are activated, parietal cells express APC functions, becoming the target of the pro-apoptotic and cytotoxic activity of crossreactive and autoreactive gastric Th1 cells. The end point of this process would be gastric corpus atrophy and hypochlorydria (Fig. 2). Cross-reactive and autoreactive gastric Th clones are able to provide B-cell help for Ig production in vitro, suggesting that chronic autoantigen-induced T-cell-dependent B-cell activation at gastric level plays a role also in the synthesis of H+, K+-ATPase autoantibodies that are usually found in the serum of AIG patients [2,4,6] and that are associated with corpus atrophy in H. pylori-infected individuals [8,9]. The relevance of cross-reactive and autoreactive Th1 effector cells in the genesis of AIG is consistent with data showing predominance of Th1 responses in other human organ-specific autoimmune diseases, such as thyroid autoimmune disorders, and multiple sclerosis. Moreover, a large body of evidence obtained in animal models suggests that in experimental organ-specific autoimmune diseases, such as encephalomyelitis, thyroiditis, gastritis, insulin-dependent diabetes mellitus, and myasthenia gravis, a pivotal pathogenic role has to be ascribed to IFN-c-secreting Th1 cells that infiltrate the target organ [23].
Almost every individual harbors autoreactive T cells, but only a few suffer from autoimmune disease. It has been proposed that pathogens might induce activation, critical expansion of autoreactive T cells and disease through autoimmune mechanisms [24]. By binding a variety of MHC class II molecules, viral and bacterial superantigens can activate large proportions of T cells, including resting autoreactive T cells, irrespective of their antigen specificity [25]. Pathogens may induce tissue inflammation and enhanced processing/presentation of self-antigens by locally activated APCs, with consequent T-cell priming. T-cell activation would then result in expansion of T cells with additional specificities (epitope spreading) [26]. Pathogens can start autoimmune disease by inducing paracrine secretion of T-cell growth factors that induce the expansion of activated autoreactive T cells to a certain threshold number that becomes sufficient to set up the disease (bystander activation) [27]. Finally, a microbial antigen can include an epitope that is structurally similar to (and cross-reactive with) an autoantigen epitope, providing the basis of molecular mimicry [28]. A clear example of mimicry in human diseases is Lyme arthritis, in which Borrelia burgdorferi disseminates to multiple body tissues. In the synovia of patients with particular MHC class II haplotypes, activation of Th1 cells reactive to the 165–173 peptide of the outer surface protein A (OspA) of B. burgdorferi occurs [29]. Such an OspA epitope is similar to the L332–340 peptide of the human leukocyte functionassociated antigen 1a (LFA-1a), whose expression is upregulated on synoviocytes by the Th1-derived IFN-c. Th1 cells specific for OspA of B. burgdorferi dominate the immune scenario in the synovial fluid of patients with Lyme arthritis, where they persist in the case of failure of antibiotic treatment. Therefore, such Th1 cells can be considered the effectors of an autoimmune process. With regard to the question of whether H. pylori is the one that starts autoimmune disease in the stomach, different hypotheses can be considered (Fig. 3). H. pylori infection may simply represent an epiphenomenon, playing no relevant role in the natural history of AIG. An alternative is that AIG patients may already have undiagnosed subclinical autoimmune disease due to inheritance of MHC haplotypes that predispose to organ-specific autoimmunity or to an altered function of regulatory T cells [30]. More likely, H. pylori infection, by providing epitopes cross-reactive to H+, K+ATPase, may lead to the expansion of both cross-reactive and autoreactive gastric T cells that are subsequently responsible for the Th1-mediated inflammation. The outcome is increased parietal cell destruction and gastric atrophy. H. pylori indeed may be the initiating factor of gastric autoimmunity through the activation of Th1 cells reactive to H. pylori peptides that cross-react with H+, K+-ATPase. This would lead to an inflammatory process in which T-cell-derived IFN-c allows H+, K+-ATPase-bearing parietal cells to act as APCs, which then undergo cell death either by activation of
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in the previous decades might have harbored the bacterium. H. pylori would then be lost, while mucosal atrophy was ongoing. The possibility that H. pylori is lost due to increasing gastric atrophy has been reported [1,6]. A broad repertoire of culprit epitopes was identified in both the pathogen and in the gastric autoantigen associated with AIG. The H. pylori cross-reactive peptides, like self H+, K+-ATPase epitopes and the entire autoantigen, were able to elicit vigorous responses by gastric Th cells. Cross-reactive Th cells quantitatively represented a significant component of the T-cell gastric infiltrate in the course of the autoimmune disease and the concomitant H. pylori infection [6]. This would argue against the possibility that the detection at gastric level of autoreactive, cytotoxic, and pro-apoptotic Th1 cells that cross-react to H. pylori epitopes is simply an epiphenomenon. Thus, in genetically susceptible individuals, H. pylori infection would trigger the development of gastric autoimmunity via molecular mimicry. This concept can have practical implications for the management of H. pylori infection and the prevention of gastric atrophy and gastric cancer.
Acknowledgments We thank the Associazione Italiana per la Ricerca sul Cancro (AIRC), the Ministry of Health, the Ministry of University and Scientific Research (MIUR), the Istituto Superiore di Sanità, and the Netherlands Organization for Scientific Research (NWO) for their financial support to our studies. Fig. 3. Factors potentially involved in the genesis of human gastric autoimmunity (AIG).
their apoptotic program or becoming the target of perforinmediated killing by Th1 cells. Apoptotic parietal cells would then allow cross-priming of T cells specific for “private” H+, K+-ATPase epitopes, ultimately leading to full-blown AIG by epitope spreading.
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[18] M.M. D’Elios, B.J. Appelmelk, A. Amedei, M.P. Bergman, G. Del Prete, Gastric autoimmunity: the role of Helicobacter pylori and molecular mimicry, Trends Mol. Med. 10 (2004) 316–323. [19] M.M. D’Elios, M. Manghetti, M. De Carli, F. Costa, C.T. Baldari, D. Burroni, et al., Th1 effector cells specific for Helicobacter pylori in the gastric antrum of patients with peptic ulcer disease, J. Immunol. 158 (1997) 962–967. [20] K.W. Wucherpfennig, A. Sette, S. Southwood, C. Oseroff, M. Matsui, J.L. Strominger, et al., Structural requirements for binding of an immunodominant myelin basic protein peptide to DR2 isotypes and for its recognition by human T cell clones, J. Exp. Med. 179 (1994) 279–290. [21] K. Bachmaier, N. Neu, L.M. De la Maza, S. Pal, A. Hessel, J.M. Penninger, Chlamydia infections and heart disease linked through antigenic mimicry, Science 283 (1999) 1335–1339. [22] A.C. Marshall, F. Alderuccio, B.H. Toh, Fas/CD95 is required for gastric mucosal damage in autoimmune gastritis, Gastroenterology 123 (2002) 780–789. [23] M.M. D’Elios, G. Del Prete, Th1/Th2 cytokine network, in: G. Martino, L. Adorini (Eds.), Topics in Neuroscience, Springer-Verlag, Berlin, 1999, pp. 68–82. [24] K.W. Wucherpfennig, Mechanisms for the induction of autoimmunity by infectious agents, J. Clin. Invest. 108 (2001) 1097–1104. [25] M.T. Schrer, L. Ignatowicz, G.M. Winslow, L.W. Kappler, P. Marrack, Superantigens: bacterial and viral proteins that manipulate the immune system, Annu. Rev. Cell Biol. 9 (1993) 101–128. [26] P.V. Lehmann, T. Forsthuber, A. Miller, E.E. Sercarz, Spreading of T-cell autoimmunity to cryptic determinants of an autoantigen, Nature 358 (1992) 155–157. [27] K. Murali-Krishna, J.D. Altman, M. Suresh, D.J. Sourdive, A.J. Zajac, J.D. Miller, et al., Counting antigen-specific CD8 T cells: a reevaluation of bystander activation during viral infection, Immunity 8 (1998) 177–187. [28] J.A. Lori, R.D. Inman, Molecular mimicry and autoimmunity, N. Engl. J. Med. 341 (1999) 2068–2074. [29] D.M. Gross, T. Forsthuber, M. Tary-Lehmann, C. Etling, K. Ito, Z.A. Nagy, et al., Identification of LFA-1 as a candidate autoantigen in treatment-resistant Lyme arthritis, Science 281 (1998) 703–706. [30] E.M. Shevach, Regulatory T cells in autoimmunity, Annu. Rev. Immunol. 18 (2000) 423–449. [31] N.R. Rose, C. Bona, Defining criteria for autoimmune diseases (Witebsky’s postulates revisited), Immunol. Today 14 (1993) 426–430.
Review
Helicobacter pylori and gastric autoimmunity Mario Milco D’Elios a,*, Mathijs P. Bergman b,c, Amedeo Amedei a, Ben J. Appelmelk b, Gianfranco Del Prete a a Department of Internal Medicine, University of Florence, Viale Morgagni, 85-50134 Florence, Italy Department of Medical Microbiology, Vrije Universiteit, Medical School, van der Boechorstraat 7, 1081 BT Amsterdam, The Netherlands c Department of Medical Microbiology and Infectious Diseases, Erasmus Medical Center, Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands b
Available online 05 November 2004
Abstract Host specific T-cell response is critical for the outcome of Helicobacter pylori infection. In genetically susceptible individuals, H. pylori can activate gastric CD4+ Th1 cells that recognize cross-reactive epitopes shared by H. pylori proteins and self H+, K+-ATPase, leading to gastric autoimmunity via molecular mimicry. © 2004 Elsevier SAS. All rights reserved. Keywords: Autoimmunity; Mimicry; Mucosal immunity; Th1–Th2 response; Cytokines; Helicobacter pylori; Atrophic gastritis; Apoptosis; H+, K+-ATPase
1. Introduction Helicobacter pylori is a Gram-negative bacterium that infects half the world’s population. H. pylori infection may remain asymptomatic lifelong or result in different clinical outcomes, such as chronic active gastritis, peptic ulcer, mucosal atrophy, gastric cancer, or gastric B-cell lymphoma of the mucosa-associated lymphoid tissue (MALT) [1]. Autoimmune—type A—gastritis (AIG) is an organspecific inflammatory disease, which usually does not result in overt symptoms until development of gastric atrophy or pernicious anemia (PA). AIG gastritis, which involves the gastric corpus and fundus, but not the antrum, is characterized by (a) serum autoantibodies to gastric H+, K+-ATPase, (b) high serum gastrin levels, (c) decreased acid secretion, (d) decreased pepsinogen I/II ratio and (e) frequent association with thyroid or other endocrine autoimmune disorders [2]. Gastric atrophy, characterized by loss of corpus and antrum glands, is considered a precursor of gastric adenocarcinoma. Since H. pylori increases the risk of gastric adenocarcinoma, it has been classified as a class I oncogenic factor [3]. The mechanisms involved in the development of mucosal atrophy are still unknown. H. pylori-related atrophic gastritis and * Corresponding author. Tel.: +39-05-5429-6445; fax: +39-05-5427-1494. E-mail address: [email protected] (M.M. D’Elios). 1286-4579/$ - see front matter © 2004 Elsevier SAS. All rights reserved. doi:10.1016/j.micinf.2004.10.001
AIG, which share a number of clinical, epidemiological and immunological characteristics, have recently been the matter of investigation [4–6]. This article will focus on the experimental evidence suggesting that H. pylori infection is involved in the genesis of gastric autoimmunity via molecular mimicry and the activation of T cells that mediate gastric damage.
2. H. pylori infection, gastric autoantibodies and atrophic gastritis Half of H. pylori-infected individuals (range 49–64%) have serum autoantibodies reactive to gastric parietal cells [7,8]. H. pylori colonization takes place in the antrum, while infected humans and experimentally infected mice develop atrophy in the corpus. The mechanisms involved in the genesis of H. pylori-associated atrophic gastritis are still poorly known. In humans, a series of observations suggests that gastric corpus atrophy can be related to an autoimmune process driven by H. pylori. Immunohistochemistry analysis of human normal gastric tissue incubated with serum from H. pylori-infected patients revealed that two different sites of gastric mucosa are targets of autoantibodies: one on the luminal membranes of the foveolar epithelial cells in the antrum and corpus mucosa, and the other one on the canalicular membranes of parietal cells in the gastric corpus mu-
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cosa [8]. Parietal cells secrete gastric acid, and their apical secretory canaliculi are very rich in H+, K+-ATPase, the gastric proton pump. The gastric H+, K+-ATPase has been identified as the major target autoantigen in chronic H. pylori gastritis with corpus atrophy [9]. Sera of H. pylori-infected mice cross-react with the human gastric mucosa, and these cross-reactive antibodies can be removed by pre-incubation with H. pylori cells because lipopolysaccharide (LPS) of H. pylori expresses Lewis blood group (Le) antigens similar to those expressed on human cells, including gastric epithelial cells [10]. In mice, H. pylori infection can induce autoantibodies through mimicry of Lewis antigens on the gastric proton pump [11]. H. pyloriinfected mice develop antibodies to H. pylori LPS, to Lewis x (Lex) and Ley and to murine H+, K+-ATPase. In mice, Lex and Ley are expressed on gastric mucin, whereas Ley is expressed on the b-subunit of gastric H+, K+-ATPase. In humans, however, serum autoantibodies recognize both the native and the recombinant H+, K+-ATPase lacking Lewis antigens [9], and they cannot be removed by pre-incubation with H. pylori cells [11]. Therefore, human anti-H+, K+ATPase autoantibodies associated with H. pylori infection do not involve Lewis mimicry, as it is for Helicobacter infection in mice [12]. Both antiluminal and anticanalicular antibodies are significantly associated with H. pylori infection. However, in H. pylori-induced atrophic gastritis, only anticanalicular autoantibodies positively correlate with clinical parameters and severity of H. pylori gastritis in the corpus, as well as with atrophy and increased corpus apoptosis [8,13]. The a- and b-subunits of H+, K+-ATPase are the targets of anticanalicular autoantibodies, and patients with H+, K+ATPase autoreactivity show more severe mucosal atrophy [9]. In addition, H. pylori-infected patients with anticanalicular autoantibodies have significantly higher serum gastrin levels and lower pepsinogen I:II ratio, which are considered markers for corpus atrophy [8,14]. The presence of anticanalicular autoantibodies correlates with decreased gastric acid output in non-ulcer dyspepsia patients infected with H. pylori [14]. On the basis of the similar histopathological and clinical characteristics of H. pylori-associated atrophic gastritis with anticanalicular antibodies and classical AIG, it has been suggested that H. pylori can represent a causative agent of gastric autoimmunity [11]. In support of this hypothesis are the observations that after H. pylori eradication, gastric autoantibodies decrease, and histologically defined AIG can be successfully reversed in most infected patients [15,16]. However, individuals with severe long-lasting atrophic corpus gastritis do not have amelioration of atrophy or intestinal metaplasia after cure of H. pylori infection, though achieving increased gastric acid secretion and reduced hypergastrinemia [17]. A strong association between H. pylori infection and gastric autoimmunity is supported by studies indicating that most patients with AIG have or have had H. pylori infection. Actual H. pylori colonization could be detected by histology
in only 10–14% of PA patients and 33% of individuals with atrophic corpus gastritis, and a single study reported even a negative association between H. pylori and PA. The conflicting prevalence of H. pylori infection in AIG/PA can be largely ascribed to the methods (histology/breath test vs. serology) used for defining H. pylori infection. Serum antibodies to H. pylori were measured in 51–83% of patients with PA and in 53–86% of patients with atrophic corpus gastritis, providing indirect support to the concept that a remarkable proportion of patients with atrophic gastritis have had H. pylori gastritis before the pathogen was cleared by the development of hypochlorydria and atrophy (reviewed in [18]).
3. T-cell responses in H. pylori-infected patients with AIG H. pylori-associated atrophic gastritis is characterized by an inflammatory infiltrate in the gastric mucosa, including T cells, macrophages and B cells. It mainly affects the corpus and the fundus, like classical AIG, and time after time it is accompanied by loss of gastric parietal and zymogenic cells. In the last few years, we characterized at the molecular level the gastric T-cell-mediated responses to H. pylori and to the H+, K+-ATPase autoantigen in a series of H. pylori-infected patients with gastric autoimmunity [6]. The clonal progenies of in vivo activated gastric T cells recovered from gastric biopsies were screened for their ability to proliferate in response to H. pylori and to H+, K+ATPase. Among gastric CD4+ helper T-cell (Th) clones, a number proliferated to H. pylori, but not to the recombinant H. pylori proteins CagA, VacA, hsp, urease nor to H+, K+ATPase. Some other Th clones proliferated in response to H+, K+-ATPase and not to H. pylori (autoreactive), and a third group of clones was found that proliferated to both H. pylori and H+, K+-ATPase [6] (Table 1). All the Th clones able to proliferate to H+, K+-ATPase were studied for their ability to respond to 205 overlapping 15-mer peptides covering the amino acid sequence of the a chain and 56 peptides for the b chain of the human H+, K+-ATPase. In the series of 13 clones that proliferated to both H+, K+-ATPase and H. pylori lysate, 11 recognized their epitope in the a chain, and two clones in the b chain. In the subgroup of Th clones that proliferate exclusively to H+, K+-ATPase, six recognized their epitope in the a chain and nine in the b chain of the proton pump. It is of note that a substantial proportion of those epitopes of H+, K+-ATPase are almost the same as those that are the target of autoreactive gastric T-cell response in AIG patients without evidence of concomitant H. pylori infection [4,5]. No overlap indeed was found between the H+, K+-ATPase epitopes recognized by clones reactive to H+, K+-ATPase only and the H+, K+-ATPase epitopes recognized by clones able to proliferate to both H+, K+-ATPase and H. pylori lysate (Table 1) [6]. Therefore, a number of H+, K+-ATPase epitopes can be considered as “private”, whereas other (“shared”) epitopes,
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Table 1 Differences in H+, K+-ATPase epitope specificities between cross-reactive and autoreactive gastric Th clones from H. pylori-infected AIG patients Th clones reactive to both H+, K+-ATPase and H. pylori (cross-reactive) H+, K+-ATPase ␣ chain peptides recognized PA.P24 (Vb4) a46–60 FF.A15 (Vb18) a181–195 FF.C32 (Vb16) a241–255 FF.C27 (Vb5.2) a256–270 FF.C26 (Vb21.3) a516–530 GR.A12 (Vb19) a576–590 GR.C31 (Vb13) a621–635 FF.A05 (Vb16) a621–635 GR.A04 (Vb13) a781–795 PA.R37 (Vb15) a836–850 MI.A30 (Vb13) a836–850 H+, K+-ATPase b chain peptides recognized FF.C13 (Vb14) b11–25 GR.C27 (Vb13) b216–230
T-cell clones reactive to H+, K+-ATPase (autoreactive) PA.Q08 (Vb4) GR.C11 (Vb5.3) PA.P34 (Vb19) FF.C39 (Vb9) PA.P02 (Vb8) PA.P14 (Vb19)
a1–15 a31–45 a151–165 a351–365 a881–895 a881–895
FF.A09 (Vb23) FF.A33 (Vb6.7) FF.C15 (Vb22) GR.A01 (Vb6.7) FF.C03 (Vb16) PA.R17 (Vb4) GR.C26 (Vb13) MI.A42 (Vb8) MI.B46 (Vb17)
b76–90 b76–90 b76–90 b81–95 b81–95 b111–125 b166–180 b231–245 b231–245
H+, K+-ATPase epitopes recognized by gastric Th clones derived from AIG patients with current H. pylori infection. Cross-reactive Th clones recognize some H+, K+-ATPase epitopes, whereas autoreactive Th clones recognized other H+, K+-ATPase epitopes. The gastric clones are indicated by the acronime of the donors (GR, PA, MI and FF), and the respective TCR Vß chain expression is reported.
Table 2 H. pylori and H+, K+-ATPase peptides recognized by gastric cross-reactive Th clones Th clone
GR.C31 GR.A04 PA.P24 PA.R37 MI.A30 FF.A15 FF.C32 FF.C27 FF.C26 FF.A05
Amino acid sequences recognized H. pylori cross-reactive peptide H+, K+-ATPase epitope VRVDVRRLDHLMNLI IRVIMVTGDHPITAK ISNLPYYIATRLVLN NLKKSIAYTLTKNIP LNNYQKENSLYNHNL KKEMEINDHQLSVAE NMRVFIIHLSPKTCK KAESDIMHLRPRNPK NMRVFIIHLSPKTCK KAESDIMHLRPRNPK VVQGGDKFHAPVLVD VIRDGDKFQINADQL VIQIGPMPTPAIAFL CTHESPLETRNIAFF ALDSLEKVVARLVVK STMCLEGTAQGLVVN VFKGIPGLSLEAVEK VMKGAPERVLERCSS IRIVKTTGDKILDAP IRVIMVTGDHPITAK
Amino acid sequence position H. pylori protein autoantigen 264–278 histidine kinase 621–635 a H+, K+-ATPase 99–113 dimethyl adenosine transferase 781–795 a H+, K+-ATPase 104–118 penicillin-binding protein 2 46–60 a H+, K+-ATPase 11–25 LPS biosynthesis protein 836–850 a H+, K+-ATPase 11–25 LPS biosynthesis protein 836–850 a H+, K+-ATPase 93–107 acetate kinase 181–195 a H+, K+-ATPase 70–84 phosphoglucosamine mutase 241–255 a H+, K+-ATPase 78–92 virb4 homolog 256–270 a H+, K+-ATPase 571–585 glucose-inhibited division pA 516–530 a H+, K+-ATPase 35–49 porphobilinogen deaminase 621–635 a H+, K+-ATPase
For each gastric Th clone reactive to both H. pylori and H+, K+-ATPase, one H. pylori cross-reactive peptide and one H+, K+-ATPase epitope were identified. Identical amino acid residues in the cross-reactive H. pylori and H+, K+-ATPase peptides recognized are highlighted.
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Fig. 1. Human H+, K+-ATPase and H. pylori mimic T-cell epitopes identified in gastric autoimmunity.
mainly in the a chain, are cross-reactive with epitopes of H. pylori antigens.
4. T-cell-mediated mucosal damage in gastric autoimmunity
Of the cross-reactive Th clones, two recognized the a836– 850 epitope, and two other ones from different patients recognized the a621–635 epitope (Table 2). A cross-reactive H. pylori peptide could be identified for 10 H+, K+-ATPase/H. pylori cross-reactive gastric Th clones [6]. Two clones from different patients, which shared recognition of the a836– 850 H+, K+ATPase epitope, also shared cross-reactivity to the 11–25 peptide of a LPS biosynthesis protein of H. pylori (Table 2). Of two other clones from different patients, both reactive to H+, K+-ATPase a621–635, one proliferated to the H. pylori histidine kinase 264–278 peptide, whereas the other showed cross-recognition of the H. pylori porphobilinogen deaminase 35–49 epitope. Overall, that study led to the identification of nine H. pylori proteins, each harboring a T-cell peptide suitable for cross-reaction with T-cell epitopes of gastric H+, K+-ATPase a chain (Fig. 1). Interestingly, none of the bacterial epitopes recognized by cross-reactive T-cell clones belong to the known H. pylori immunodominant antigens, such as CagA, VacA and urease, which are major targets of gastric T-cell responses in H. pylori-infected patients with peptic ulcer [19]. The sequence homology between cross-reactive H. pylori and H+, K+-ATPase peptides varies and can be more or less stringent (Table 2), considering that degeneracy in both TCR and MHC binding motifs can reduce sequence-specific requirement to only a few crucial residues [20]. A clear example is provided by the animal model of myocarditis, where cross-recognition of the autoantigen (e.g. myosin) and a chlamydial peptide depends only on four identical residues in the amino acid sequence [21].
Upon stimulation with the relevant peptide, all crossreactive Th clones and most of the H+, K+-ATPase-specific Th clones from the gastric corpus of H. pylori-infected AIG patients produced IFN-c, but not IL-4 (Th1 cytokine profile) [6]. All the cross-reactive and autoreactive H+, K+-ATPasespecific clones also produced high concentrations of TNF-a. Production of IFN-c, and TNF-a by Th1 cells can locally act, increasing the expression by gastric epithelial cells of both MHC class II molecules and B7-1 (CD80) or B7-2 (CD86) costimulatory molecules, as well as cathepsins involved in antigen processing, thus favoring the presentation of peptides by such non-professional antigen-presenting cells (APCs) [18]. Following activation, virtually all gastric cross-reactive and autoreactive Th clones were able to induce cell death via either Fas–Fas ligand-mediated apoptosis or perforinmediated cytotoxicity against target cells which they were in close contact with [4,6]. Parietal cells can express Fas (CD95) molecule, and HLA-DR is ectopically expressed on glandular epithelium in proximity to T-cell infiltrates in human AIG [12,18]. Requirement for Fas in development of full-blown experimental autoimmune gastritis (EAIG) has been demonstrated in the model of lpr/lpr mice, which are deficient in Fas expression and fail to develop destructive gastritis and autoantibody production upon neonatal thymectomy [22]. Up-regulated Fas expression by gastric parietal cells in EAIG mice may explain, at least in part, the selective destruction of parietal cells [22]. Based on these observations, it is tempting to speculate that in the inflammatory scenario in which cross-reactive and autoreactive Th clones
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5. What gives the start to gastric autoimmunity?
Fig. 2. Model of T-cell-mediated damage in H. pylori infection with gastric autoimmunity. H. pylori induces in susceptible individuals the expansion of H. pylori-specific Th1 cells that cross-react with H+, K+-ATPase epitopes. H. pylori-H+, K+-ATPase cross-reactive Th1 cells could mediate destruction of gastric mucosa, via Fas-ligand (Fas-L)-induced apoptosis and perforinmediated cytotoxicity.
are activated, parietal cells express APC functions, becoming the target of the pro-apoptotic and cytotoxic activity of crossreactive and autoreactive gastric Th1 cells. The end point of this process would be gastric corpus atrophy and hypochlorydria (Fig. 2). Cross-reactive and autoreactive gastric Th clones are able to provide B-cell help for Ig production in vitro, suggesting that chronic autoantigen-induced T-cell-dependent B-cell activation at gastric level plays a role also in the synthesis of H+, K+-ATPase autoantibodies that are usually found in the serum of AIG patients [2,4,6] and that are associated with corpus atrophy in H. pylori-infected individuals [8,9]. The relevance of cross-reactive and autoreactive Th1 effector cells in the genesis of AIG is consistent with data showing predominance of Th1 responses in other human organ-specific autoimmune diseases, such as thyroid autoimmune disorders, and multiple sclerosis. Moreover, a large body of evidence obtained in animal models suggests that in experimental organ-specific autoimmune diseases, such as encephalomyelitis, thyroiditis, gastritis, insulin-dependent diabetes mellitus, and myasthenia gravis, a pivotal pathogenic role has to be ascribed to IFN-c-secreting Th1 cells that infiltrate the target organ [23].
Almost every individual harbors autoreactive T cells, but only a few suffer from autoimmune disease. It has been proposed that pathogens might induce activation, critical expansion of autoreactive T cells and disease through autoimmune mechanisms [24]. By binding a variety of MHC class II molecules, viral and bacterial superantigens can activate large proportions of T cells, including resting autoreactive T cells, irrespective of their antigen specificity [25]. Pathogens may induce tissue inflammation and enhanced processing/presentation of self-antigens by locally activated APCs, with consequent T-cell priming. T-cell activation would then result in expansion of T cells with additional specificities (epitope spreading) [26]. Pathogens can start autoimmune disease by inducing paracrine secretion of T-cell growth factors that induce the expansion of activated autoreactive T cells to a certain threshold number that becomes sufficient to set up the disease (bystander activation) [27]. Finally, a microbial antigen can include an epitope that is structurally similar to (and cross-reactive with) an autoantigen epitope, providing the basis of molecular mimicry [28]. A clear example of mimicry in human diseases is Lyme arthritis, in which Borrelia burgdorferi disseminates to multiple body tissues. In the synovia of patients with particular MHC class II haplotypes, activation of Th1 cells reactive to the 165–173 peptide of the outer surface protein A (OspA) of B. burgdorferi occurs [29]. Such an OspA epitope is similar to the L332–340 peptide of the human leukocyte functionassociated antigen 1a (LFA-1a), whose expression is upregulated on synoviocytes by the Th1-derived IFN-c. Th1 cells specific for OspA of B. burgdorferi dominate the immune scenario in the synovial fluid of patients with Lyme arthritis, where they persist in the case of failure of antibiotic treatment. Therefore, such Th1 cells can be considered the effectors of an autoimmune process. With regard to the question of whether H. pylori is the one that starts autoimmune disease in the stomach, different hypotheses can be considered (Fig. 3). H. pylori infection may simply represent an epiphenomenon, playing no relevant role in the natural history of AIG. An alternative is that AIG patients may already have undiagnosed subclinical autoimmune disease due to inheritance of MHC haplotypes that predispose to organ-specific autoimmunity or to an altered function of regulatory T cells [30]. More likely, H. pylori infection, by providing epitopes cross-reactive to H+, K+ATPase, may lead to the expansion of both cross-reactive and autoreactive gastric T cells that are subsequently responsible for the Th1-mediated inflammation. The outcome is increased parietal cell destruction and gastric atrophy. H. pylori indeed may be the initiating factor of gastric autoimmunity through the activation of Th1 cells reactive to H. pylori peptides that cross-react with H+, K+-ATPase. This would lead to an inflammatory process in which T-cell-derived IFN-c allows H+, K+-ATPase-bearing parietal cells to act as APCs, which then undergo cell death either by activation of
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in the previous decades might have harbored the bacterium. H. pylori would then be lost, while mucosal atrophy was ongoing. The possibility that H. pylori is lost due to increasing gastric atrophy has been reported [1,6]. A broad repertoire of culprit epitopes was identified in both the pathogen and in the gastric autoantigen associated with AIG. The H. pylori cross-reactive peptides, like self H+, K+-ATPase epitopes and the entire autoantigen, were able to elicit vigorous responses by gastric Th cells. Cross-reactive Th cells quantitatively represented a significant component of the T-cell gastric infiltrate in the course of the autoimmune disease and the concomitant H. pylori infection [6]. This would argue against the possibility that the detection at gastric level of autoreactive, cytotoxic, and pro-apoptotic Th1 cells that cross-react to H. pylori epitopes is simply an epiphenomenon. Thus, in genetically susceptible individuals, H. pylori infection would trigger the development of gastric autoimmunity via molecular mimicry. This concept can have practical implications for the management of H. pylori infection and the prevention of gastric atrophy and gastric cancer.
Acknowledgments We thank the Associazione Italiana per la Ricerca sul Cancro (AIRC), the Ministry of Health, the Ministry of University and Scientific Research (MIUR), the Istituto Superiore di Sanità, and the Netherlands Organization for Scientific Research (NWO) for their financial support to our studies. Fig. 3. Factors potentially involved in the genesis of human gastric autoimmunity (AIG).
their apoptotic program or becoming the target of perforinmediated killing by Th1 cells. Apoptotic parietal cells would then allow cross-priming of T cells specific for “private” H+, K+-ATPase epitopes, ultimately leading to full-blown AIG by epitope spreading.
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