Cross-reaction of antibody against Helicobacter pylori ... - Springer Link

Int J Hematol (2009) 89:142–149 DOI 10.1007/s12185-008-0247-4

ORIGINAL ARTICLE

Cross-reaction of antibody against Helicobacter pylori urease B with platelet glycoprotein IIIa and its significance in the pathogenesis of immune thrombocytopenic purpura Yanyan Bai Æ Zhaoyue Wang Æ Xia Bai Æ Ziqiang Yu Æ Lijuan Cao Æ Wei Zhang Æ Changgeng Ruan

Received: 22 August 2008 / Accepted: 17 December 2008 / Published online: 30 January 2009 Ó The Japanese Society of Hematology 2009

Abstract Many clinical investigations have suggested that Helicobacter pylori (H. pylori) infection might be associated with immune thrombocytopenic purpura (ITP), but its role in the pathogenesis of ITP is unsettled. In this study, we cultured H. pylori, produced recombinant H. pylori urease (ure) B, and then prepared monoclonal antibody (MoAb) against ureB, 1F11, both 1F11 and MoAb against human platelet glycoprotein (GP) IIIa, SZ21, could bind to the band of GP IIIa of normal platelet lysate, but not to that from a patient with Glanzmann thrombasthenia (GT) whose GP IIb–IIIa complex was absent. Flow cytometry showed that normal platelets were reacted with 1F11 and SZ21, while GT platelets were not. In immuno-radiometric assay, the binding of 125I-labeled 1F11 to GP IIIa was higher than that to GP Ib, GP IIb, GP VI, and P-selectin. 1F11 could partly compete with SZ21 in a binding to platelet surface. In addition, 1F11 inhibited platelet aggregation induced by adenosine diphosphate, but had no effect on platelet P-selectin expression or Thromboxane B2 production of platelets. These results indicate that H. pylori ureB antibody could cross-react with human platelet GP IIIa and partly inhibit platelet aggregation. UreB may be a crucial component of H. pylori involved in the pathogenesis of a subset of ITP.

Electronic supplementary material The online version of this article (doi:10.1007/s12185-008-0247-4) contains supplementary material, which is available to authorized users. Y. Bai Z. Wang (&) X. Bai Z. Yu L. Cao W. Zhang C. Ruan Jiangsu Institute of Hematology, The First Affiliated Hospital of Soochow University, Suzhou, China e-mail: [email protected]

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Keywords Helicobacter pylori Urease Platelet Glycoprotein Cross-reaction

1 Introduction Immune thrombocytopenic purpura (ITP) is a common bleeding disorder caused by autoantibody-mediated thrombocytopenia. However, the factors that incite this disorder remain uncertain, though some infectious agents have been thought to be responsible for the formation of platelet antibodies. In 1984, Marshall and Warren [1] discovered Helicobacter pylori (H. pylori) and deciphered its role in gastritis and peptic ulcer disease. Following this paradigm discovery, several studies suggested that H. pylori infection may be associated with such extragastric diseases as rheumatoid arthritis [2], Sjo¨gren’s syndrome [3], systemic lupus erythematosus [4], iron deficiency anemia [5] and mucosa-associated lymphoid tissue lymphoma [6]. In addition, Gasbarrini et al. [7] first reported 8 of 11 H. Pylori-positive patients with ITP experienced significant increase in the platelet count following their H. Pylori had been eradicated. After that, several reports also showed efficacy of H. pylori eradication in raising platelet count in ITP patients [8–11]. However, other studies did not support a role for H. pylori in ITP [12, 13]. The reasons for these inconsistent outcomes are uncertain, and the mechanism by which H. pylori infection might contributes to the development of ITP remains to be elucidated. Many interpretations could be considered, including the possibility that there might exist antigen mimicry between H. pylori and platelet glycoproteins (GP). On the other hand, many genetically diverse H. pylori strains with different antigens and virulence have been found, which could partly explain the striking discrepancies between the reported results [14].

Cross-reaction of antibody against Helicobacter pylori urease B

Although large chromosomal regions and most genes vary from H. pylori strain to strain, urease (ure) B is very well conserved (97–100% amino acid identity, based on BlastP analysis of GenBank sequences), and is central to the pathogenesis of the bacterium [15]. The major antigenic component for antibody production against H. pylori is its ure [16], and it has been suggested that H. pylori components, ureB in particular, could initiate various autoimmune diseases via producing autoreactive antibodies through the activation of B-1 cells [17]. We therefore conducted a study to assess the role of H. pylori ureB in the pathogenesis of ITP and to investigate its immune mechanisms.

2 Materials and methods 2.1 Bacterial growth conditions The preparation of tissue and blood samples was approved by the Board of Research Ethics of our hospital, and informed written consent was obtained from each participant in accordance with the Declaration of Helsinki. Bacteria H. pylori were isolated from an antral biopsy sample of a patient with peptic ulcer presenting for gastroscopy, and then cultured on H. pylori selective agar plates with 5% defibrinated sheep blood and antibiotics at 37°C under microaerophilic conditions with 5% O2, 15% CO2 and 80% N2 following methods as described previously [18]. After being cultured for 5 days, the colonies were scraped and stained to confirm that the cultured bacilli were Gram-negative, flagellate or curved H. pylori (Fig. 1).

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2.2 Construction of recombinant expression plasmid pQE30-ureB Genomic DNA of H. pylori was extracted with phenol/ chloroform/isopentanol, precipitated with ethanol, and then stored at -20°C. H. pylori ureB gene was amplified by polymerase chain reaction (PCR) using a primer set which were designed according to the complete DNA sequence of H. pylori published, containing BglII site in P1 and XhoI site in P2, respectively (P1: 50 -GAAAGATCTAAAAAG ATTAGCAGAAAAG-30 ; P2:50 -GGCTCGAGGCAGAAA ATGCTAAAGAGTT-30 ). Amplication conditions were as follows: at 95°C for 5 min, then 35 cycles at 95°C for 30 s, at 55°C for 50 s and at 72°C for 90 s, followed by 10 min at 72°C. The PCR product was separated using a UNIQ-10 column purification kit (Biotech Co., Shanghai, China), ligated into expression plasmid pUCm-T (Biotech Co., Shanghai, China), then separated by electrophoresis on a 1% agarose gel, and stained with ethidium bromide to verify the presence of products of the expected size. The nucleotide sequence of amplified DNA fragment was subjected to direct cycle sequence analysis on an ABI PRISM 377 DNA Sequencer (Applera, Foster, CA, USA). The fragment of BamHI and SalI-digested pUCmTureB was inserted into the BamHI/SalI site of expression vector pQE30 in ligation buffer supplemented with PEG 40000 and T4DNA ligase. The recombinant pQE30-ureB plasmid was digested with restriction enzymes EcoRI and HindIII, and then electrophoretically analyzed in 1% agarose. 2.3 In vitro transformation and expression of UreB

Fig. 1 H. pylori were isolated from the biopsy sample of a patient with peptic ulcer, and cultured under a microaerophilic condition. The colony stain showed that the cultured bacilli were Gram-negative, flagellate or curved

Recombinant plasmid of pQE30-ureB was transformed into competent cells of Escherichia coli TG1 using calcium chloride. The transformed cells were cultured in LB medium (tryptone 10 g/L, yeast extract 5 g/L, NaCl 5 g/L; pH 7.4) supplemented with 100 lg/ml ampicillin and 25 lg/ml kanamycin. The cells were harvested in a refrigerated centrifuge (0°C) at 4,0009g, and then treated in ultrasound wave to remove the cellular debris. After being passed through resin column, the purified ureB protein was electrophoretically analyzed in a 12% polyacrylamide gel, transferred onto nitrocellulose membrane, and then incubated overnight with anti-6 9 His antibody, washed several times with TBST (50 mm Tris, 150 mM NaCl, 0.1% Tween-20; pH 7.5), and incubated for 2 h with horse radish peroxidase (HRP)-labeled goat-anti-mouse immunoglobulin (Ig) G (Sigma-Aldrich, Shanghai, China). The immunospecific bands were visualized by chemiluminescent substrate (ECL).

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2.4 Preparation of monoclonal antibody against H. pylori ureB

washing six times, the radioactivity of the each well was counted in the c counter.

The recombinant ureB was used to develop monoclonal antibody (MoAb) against H. pylori ureB with the classical hybridoma technique as described by Ruan et al. [19]. Briefly, hybridomas were obtained by fusing mouse myeloma cells SP2/0 with splenocytes from the mice immunized with purified H. pylori UreB. Hybridomas 1F11 secreting antibodies against H. pylori ureB were obtained and subcloned. Ascites of MoAb were prepared by injecting 1 9 106 cells of hybridoma 1F11 into mice abdomen. Ig G was purified from 1F11 ascites fluid by chromatography on protein-A-Sepharose CL-4B. The purified 1F11 was separated on 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) under reducing conditions to verify the two bands of heavy chain and light chain with molecular weights of 55,000 and 23,000, respectively. Meanwhile SDS–PAGE and Western blot analysis of the lysate of H. pylori bacteria was performed as described above. The transferred proteins were incubated with MoAb 1F11, and then with HRP-goat-anti-mouse Ig G. The immunospecific bands were visualized by ECL.

2.6 Influence of 1F11 on platelet aggregation and activation

2.5 Cross-reaction of H. pylori ureB antibody with human platelets Platelet-rich plasma (PRP) from normal individuals and two patients with Glanzmann thrombasthenia (GT) was prepared from 10 mL of 3.8% (w/v) sodium citrate blood (blood/ anticoagulant: 9/1). The Triton X-100 lysate of H. pylori or platelets was prepared for electrophoresis and Western blotting, and then incubated with 1F11 or SZ21 as the first antibodies, and then with HPR-goat-anti-mouse antibody as the second antibody, and finally visualized by ECL. In flow cytometry (FCM) analysis of platelets, the PRP was adjusted to 109 platelets/mL. Samples of 100 lL were incubated with saturating amounts of SZ21, 1F11 or control mouse IgG, washed twice, and then incubated with saturating amount of fluorescein isothiocyanate (FITC)conjugated goat-anti-mouse IgG, and finally analyzed with an Epics-XL flow cytometer (Coulter, Miami, FL, USA) with argon laser excitation at 488 nm. Platelets were gated by FSC/SSC-characteristics. In immuno-radiometric assay (IRMA), 1F11 was radioiodinated by the chloramine T method. The flatbottomed microtiter plates were coated with MoAbs SZ2 (against GP Ib), SZ21, SZ22 (against GP IIb), SZ51 (against P-selectin) and SZ118 (against GP VI), respectively, overnight at 4°C, then blocked with 1% BSA-PBS overnight at 4°C. One hundred microliters of platelet lysate was added to each well and incubated at 37°C for 2 h, and then 125I-labeled 1F11 was added for 2 h. After

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Platelet aggregation was performed in PRP (3 9 108 platelets/mL, final concentration) using an aggregometer (Chronolog Co., Haverton, PA, USA). A series of concentrations (12.5–400 lg/mL) of MoAb 1F11 was added 10 min before the addition of 2 lmol/L adenosine diphosphate (ADP). The aggregation rates were measured according turbidimetry. The samples of aggregated PRP were centrifugated, and the P-selectin level of the supernatant was assayed using ELISA [20]. The kit was provided by Jiangsu Institute of Hematology, China. Briefly, the flat-bottomed microtiter plates were coated with 100 lL anti-P-selectin MoAb SZ51 (10 lg/mL) overnight at 4°C. One hundred microliters of standard (2.5–80 ng/mL) or supernatant was added to each well and incubated at 37°C for 2 h. After washing three times, 100 lL of another anti-P-selectin MoAb, S12, which had been conjugated to HRP, was added. The presence of P-selectin was revealed with 200 lL of o-phenylene (1 mg/mL) containing 0.03% H2O2 as the substrate. Absorbance at 405 nm was measured and the concentration of supernatant P-selectin was determined using the standard curve. Thromboxane (TX) B2 of the supernatant was also assayed using an enzyme immunoassay kit (Jiangsu Institute of Hematology, China). The microtiter plates were

Fig. 2 Recombinant expression plasmid pQE-ureB was digested by restriction enzymes EcoRI and HindIII. UreB gene of 1,700 bp was identified on agarose gel. Lane 1 pQE30-ureB; lane 2 pQE30-ureB digested by restriction enzymes EcoRI and HindIII; lane 3 DNA marker


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Fig. 3 Recombinant ureB protein of Mr 66 kDa was analyzed by SDS–PAGE and Western blot. a Lane 1 purified protein ureB; lane 2 M15 cultured supernate before induced by IPTG; lane 3 and 4 M15 cultured supernate after 4-h induced by IPTG; lane 5 marker. b ureB protein transferred on nitrocellulose membrane

Fig. 4 Identification of monoclonal antibody against H. pylori ureB, 1F11 was performed on SDS–PAGE and Western blotting. a Lane 1 and 2 two bands of heavy chain and light chain of 1F11 with molecular weights of 55,000 and 23,000, respectively, on 12% under reducing conditions; lane 3 molecular weight marker. b 1F11 specifically recognizing H. pylori ureB protein (Mr 66 kDa) on Western blotting. Lane 1 negative control; lane 2 and 3 McAb 1F11

concentration of supernatant TXB2 was determined using the standard curve as described above. 2.7 Statistical analysis

Fig. 5 Western blot analysis showed binding of 1F11 and SZ21 to the band of GP IIIa of normal platelet lysate, but not to that of platelet lysate prepared from a GT patient whose platelet membrane GP IIb/ IIIa complex was absent. Lane 1 negative control; lane 2 proteins of H. pylori reacted with 1F11 as positive control; lane 3 proteins of GP IIb/IIIa deficient platelets reacted with 1F11; lane 4 proteins of normal human platelets reacted with 1F11; lane 5 proteins of GP IIb/ IIIa deficient platelets reacted with SZ21; lane 6 proteins of normal human platelets reacted with SZ21

coated with 200 lL TXB2-BSA. After washing, 100 lL of TXB2 standard (12.5–1,600 pg/mL) or supernatant was added, then anti TXB2-IgG, HRP-goat-anti-rabbit IgG were subsequently added, and colored with o-phenylene. The

All the values were expressed as the mean ± standard deviation. Statistical analyses were performed using the SPSS 11.0 statistical software package for Windows (SPSS Inc., Chicago, IL, USA). Comparison of average values was performed by the Student’s t test. 3 Results 3.1 Constructure and analysis of recombinant plasmid pQE-ureB When recombinant plasmid pQE-ureB was digested by restriction enzymes EcoRI and HindIII, an expected

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Fig. 6 Flow cytometry analysis showed the reactivity of the normal platelets with both 1F11 and SZ21, but a low reactivity of GT patient platelets with the two McAbs. a–c Normal platelets were reacted with both 1F11 and SZ21. a Negative control; b normal human platelets

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reacted with SZ21; c normal human platelets reacted with 1F11. d–f GT patient platelets had a low reactivity with 1F11 and SZ21. d Negative control; e GT patient platelets reacted with SZ21; f GT patient platelets reacted with 1F11

react with serum from all ten H. pylori-positive patients, but not H. pylori-negative individuals, indicating ureB antigenicity. 3.2 Identification of monoclonal antibody against H. pylori ureB The prepared Ig G of MoAb against H. pylori ureB, 1F11, had two bands of heavy chain and light chain with molecular weights of 55,000 and 23,000, respectively, on 12% SDS–PAGE under reducing conditions (Fig. 4a). Western blotting confirmed that 1F11 could specifically recognize ureB protein (Mr 66 kDa) of H. pylori (Fig. 4b). Fig. 7 IRMA showed a higher radioactivity of 125I-labeled 1F11 on microtiter plates of platelet lysate-McAb against GP IIIa (SZ21) than on those of platelet lysate binded to McAbs against GP Ib (SZ2), GP IIb (SZ22), GP VI (SZ118), or P-sectin (SZ51)

3.3 Cross-reaction of monoclonal antibody 1F11 with human platelet

1,700 bp fragment of H. pylori ureB was identified on agarose gel (Fig. 2). The nucleotide sequencing analysis showed 99% homology to the ureB sequence published in Genbank. The lysate of the E. coli transfected by plasmid pQEureB was analyzed by SDS–PAGE and Western blot. A band of Mr 66 kDa corresponding to ureB protein could be observed (Fig. 3). The recombinant ureB protein could

In Western blot analysis, both 1F11 and MoAb against human platelet membrane GP IIIa, SZ21, bound to the band of protein 95 kDa (GP IIIa) of normal platelet lysate, but not to that of platelet lysate prepared from a GT patient in whom platelet GP IIb/IIIa complex was absent (Fig. 5). FCM analysis also showed that normal platelets were reacted with both 1F11 and SZ21, whereas platelets of the GT patient showed a low reactivity with 1F11 or SZ21

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Fig. 8 McAb 1F11 inhibited FITC-SZ21 binding to platelets in FCM analysis. a The positive cell percentage of control PRP (1.0%); b PRP ? FITC-SZ21 (99.5%); c PRP ? 1F11 ? FITC-SZ21 (77.1%); d PRP ? unrelated McAb ? FITC-SZ21 (96.6%)

Fig. 9 1F11 inhibited ADPinduced platelet aggregation. a Platelet aggregation curves in the presence of 2 lM ADP with or without 1F11; b platelet aggregation inhibition by 1F11 in a dose-dependent manner

(Fig. 6). In IRMA, the radioactivity of 125I-labeled 1F11 on microtiter plates of platelet lysate-MoAb SZ21 was higher than on platelet lysate bound to other four MoAbs against GP Ib, GP IIb, GP VI, or P-selectin (Fig. 7). All these

experimental results showed that there exists a crossreaction between MoAb 1F11 and platelet GP IIIa. In addition, we investigated the effect of MoAb 1F11 on FITC-SZ21 binding to platelets by using FCM. The

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positive cell percentage of platelet GP IIIa was 99.5%, which was significantly decreased by 1F11, but not by an unrelated MoAb against CD147 (Fig. 8), indicating that 1F11 could partly compete with SZ21 in a binding to GP IIIa on the surface of platelets. 3.4 Effect of MoAb 1F11 on platelet activation induced ADP MoAb 1F11 could inhibit ADP (2 lmol/L) induced platelet aggregation in a dose-dependent manner (Fig. 9). However, 1F11 had no effect on P-selectin and TXB2 levels of PRP in the presence of ADP (Table 1).

4 Discussion Immune thrombocytopenic purpura is an immune-mediated bleeding disorder in which antibody-sensitized platelets are destroyed prematurely. ITP could be incited by various bacteria and viruses, and recently it has been proposed that H. pylori might be associated with ITP. Based on the current data of extragastric manifestations of H. pylori infection, Bohr et al. [21] believed that ITP and sideropenic anemia represent the diseases in which the pathogenic link appears to be strongest. The molecular mechanism of its pathogenesis remains unclear, but could be related to crossreactivity, that is, H. pylori and platelet probably share some epitopes. Takahashi et al. [22] reported that plateletassociated IgG from several ITP patients recognized H. pylori cytotoxin-associated gene (Cag) A protein, and that cross-reactive antibody levels decreased with improved platelet count after H. pylori eradication. They proposed that molecular mimicry by CagA antigen may be involved in the pathogenesis of H. pylori-associated ITP. Another possibility has been proposed that CagA antibodies cross-react with a peptide specifically expressed by platelets of patients with ITP [23]. However, CagA-positivity varies depending upon the geographic location, and some H. pylori strains do not harbor the CagA gene [24].

Table 1 Influence of McAb against Hp ureB on P-selectin and TXB2 production by ADP-activated platelets Group Control ADP ADP ? 50 lg/mL 1F11 ADP ? 200 lg/mL 1F11

P-selectin (ng/mL) 27.9 ± 8.3

TXB2 (pg/mL) 6.4 ± 0.7

a

10.3 ± 1.3a

a

10.5 ± 1.1a

a

10.3 ± 1.2a

130.5 ± 78.4 171.1 ± 64.4 164.7 ± 56.3

n = 11, x ± s a

Compared to control, P \ 0.01; no significant difference between ADP, ADP ? 50 lg/ml 1F11 and ADP ? 200 lg/mL 1F11

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In contrary, ureB is one of highly antigenic H. pylori proteins and is very well conserved across heterologous strains of H. pylori [25]. Therefore, we investigated the possible cross-reactivity between H. pylori ureB antibody and platelet membrane GP. Firstly, we cultured H. pylori bacteria, then produced recombinant ureB which was then successfully used to prepare a specific anti-ureB MoAb 1F11 for this study. The results of the three experiments used in this study, including SDS–PAGE, FCM and IRMA, showed that MoAb against ureB could react with GP IIIa of normal platelets, but not with GT platelets whose GP IIIa was absent. And the competition of 1F11 for anti-GP IIIa antibody binding to platelets also supported this speculation. Glycoproteins IIb–IIIa complex is a receptor for fibrinogen and is required for platelet aggregation induced by ADP and other physiologic agonists. In this study, we found that H. pylori ureB antibody inhibited ADP-induced platelet aggregation in a dose-dependent manner. However, it had no effect on platelet P-selectin expression and TXB2 production, both of which are the major markers of platelet activation. This paradoxical phenomenon can be explained by the fact that platelet P-selectin expression and TXB2 production could occur before aggregation, the ‘‘final step’’ of platelet activation, just like that found in GT in which GP IIb–IIIa is deficient, but TXB2 synthesis remains normal [26]. In this study we found that the platelet aggregation was only partly inhibited by H. pylori ureB antibody. The ability of this inhibition seems not enough to exaggerate bleeding tendency in ITP patients. On the other hand, it would be interesting to investigate whether the anti-GP IIIa antibody of ITP patients could react with H. pylori ureB antigen. Unfortunately, the amount of antiGP IIIa antibody available from individual ITP patients were too limited for us to do this research. Molecular mimicry is one way to initiate the production of autoantibodies. Although there is no great similarity of the molecular structure between ureB and platelet GP IIIa, sequence homology can be limited to two or three peptide residues positions [27]. Further study is needed to identify the binding motif of H. pylori ureB antibody to platelet GP IIIa. In summary, our results first indicate that antibody against H. pylori ureB could cross-react with human platelet GP IIIa and inhibit platelet aggregation. This study might be of importance for understanding the pathogenesis of a subset of ITP, and has an intensive value for clinical diagnosis and treatment of ITP. Acknowledgments This work was supported by a grant from the Natural Science Foundation of China (30770917). Conflict of interest statement The authors have declared that no conflict of interest exists.


References 1. Marshall BJ, Warren RM. Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet. 1984;16:1311–5. doi:10.1016/S0140-6736(84)91816-6. 2. Ishikawa N, Fuchigami T, Matsumoto T, Kobayashi H, Sakai Y, Tabata H, et al. Helicobacter pylori infection in rheumatoid arthritis: effect of drugs on prevalence and correlation with gastroduodenal lesions. Rheumatology. 2002;41:72–7. doi:10.1093/ rheumatology/41.1.72. 3. De Vita S, Ferraccioli G, Avellini C, Sorrentino D, Dolcetti R, Di Loreto C, et al. Widespread clonal B-cell disorder in Sjogren’s syndrome predisposing to Helicobacter pylori-related gastric lymphoma. Gastroenterology. 1996;110:1969–74. doi:10.1053/ gast.1996.v110.pm8964425. 4. Sawalha AH, Schmid WR, Binder SR, Bacino DK, Harley JB. Association between systemic lupus erythematosus and Helicobacter pylori seronegativity. J Rheumatol. 2004;31:1546–50. 5. Hershko C, Ianculovich M, Souroujon M. A hematologist’s view of unexplained iron deficiency anemia in males: impact of Helicobacter pylori eradication. Blood Cells Mol Dis. 2007;38:45– 53. doi:10.1016/j.bcmd.2006.09.006. 6. Blaser MJ, Atherton JC. Helicobacter pylori persistence: biology and disease. J Clin Invest. 2004;113:321–33. 7. Gasbarrini A, Franceschi F, Tartaglione R, Landolfi R, Pola P, Gasbarrini G. Regression of autoimmune thrombocytopenia after eradication of Helicobacter pylori. Lancet. 1998;352:878. doi: 10.1016/S0140-6736(05)60004-9. 8. Franchini M. Thrombotic thrombocytopenic purpura: proposal of a new pathogenic mechanism involving Helicobacter pylori infection. Med Hypotheses. 2005;65:1128–31. doi:10.1016/j. mehy.2005.06.015. 9. Veneri D, Krampera M, Franchini M. High prevalence of sustained remission of idiopathic thrombocytopenic purpura after Helicobacter pylori eradication: a long-term follow-up study. Platelets. 2005;16:117–9. doi:10.1080/09537100400015153. 10. Kodama M, Kitadai Y, Ito M, Kai H, Masuda H, Tanaka S, et al. Immune response to CagA protein is associated with improved platelet count after Helicobacter pylori eradication in patients with idiopathic thrombocytopenic purpura. Helicobacter. 2007;12:36–42. doi:10.1111/j.1523-5378.2007.00477.x. 11. Asahi A, Kuwana M, Suzuki H, Hibi T, Kawakami Y, Ikeda Y. Effects of a Helicobacter pylori eradication regimen on antiplatelet autoantibody response in infected and uninfected patients with idiopathic thrombocytopenic purpura. Haematologica. 2006;91:1436–7. 12. Michel M, Cooper N, Jean C, Frissora C, Bussel JB. Does Helicobater pylori initiate or perpetuate immune thrombocytopenic purpura? Blood. 2004;103:890–6. doi:10.1182/blood-200303-0900. 13. Jarque I, Andreu R, Llopis I, De la Rubia J, Gomis F, Senent L, et al. Absence of platelet response after eradication of Helicobacter pylori infection in patients with chronic idiopathic thrombocytopenic purpura. Br J Haematol. 2001;115:1002–3. doi:10.1046/j.1365-2141.2001.03194.x.

149 14. Arents NL, van Zwet AA, Thijs JC, Kooistra-Smid AM, van Slochteren KR, Degener JE, et al. The importance of vacA, cagA, and iceA genotypes of Helicobacter pylori infection in peptic ulcer disease and gastroesophageal reflux disease. Am J Gastroenterol. 2001;96:2603–8. doi:10.1111/j.1572-0241.2001.04104.x. 15. Hovey JG, Watson EL, Langford ML, Hildebrandt E, Bathala S, Bolland JR, et al. Genetic microheterogeneity and phenotypic variation of Helicobacter pylori arginase in clinical isolates. BMC Microbiol. 2007;7:26–40. doi:10.1186/1471-2180-7-26. 16. Fan X, Gunasena H, Cheng Z, Espejo R, Crowe SE, Ernst PB, et al. Helicobacter pylori urease binds to class II MHC on gastric epithelial cells and induces their apoptosis. J Immunol. 2000; 165:1918–24. 17. Yamanishi S, Iizumi T, Watanabe E, Shimizu M, Kamiya S, Nagata K, et al. Implications for induction of autoimmunity via activation of B-1 cells by Helicobacter pylori urease. Infect Immun. 2006;74:248–56. doi:10.1128/IAI.74.1.248-256.2006. 18. Iizumi T, Yamanishi S, Kumagai Y, Nagata K, Kamiya S, Hirota K, et al. Augmentation of Helicobacter pylori urease activity by its specific IgG antibody: implications for bacterial colonization enhancement. Biomed Res. 2005;26:35–42. doi:10.2220/ biomedres.26.35. 19. Ruan G, Du XP, Xi XD, Castaldi PA, Berndt MC. A murine antiglycoprotein Ib complex monoclonal antibody, SZ 2, inhibits platelet aggregation induced by both ristocetin and collagen. Blood. 1987;69:570–7. 20. Zhao Y, He Y, Shen W, Ruan C. Plasma P-selectin measurement using immunoassay method and its clinical significance. J Exp Hematol. 1997;5:426–8. 21. Bohr UR, Annibale B, Franceschi F, Roccarina D, Gasbarrini A. Extragastric manifestations of Helicobacter pylori infection— other Helicobacters. Helicobacter. 2007;12:S45–53. doi:10.1111/j. 1523-5378.2007.00533.x. 22. Takahashi T, Yujiri T, Shinohara K, Inoue Y, Sato Y, Fujii Y, et al. Molecular mimicry by Helicobacter pylori CagA protein may be involved in the pathogenesis of H. pylori-associated chronic immune thrombocytopenic purpura. Br J Haematol. 2004;124:91–6. doi:10.1046/j.1365-2141.2003.04735.x. 23. Franceschi F, Christodoulides N, Kroll MH, Genta RM. Helicobacter pylori and idiopathic thrombocytopenic purpura. Ann Intern Med. 2004;140:766–7. 24. Perez-Perez GL, Bhat N, Gaensbauer L, Fraser A, Taylor DN, Kuipers EL, et al. Country specific constancy by age in Cag? proportion of Helicobacter pylori infection. Int J Cancer. 1997;72:453–6. doi:10.1002/(SICI)1097-0215(19970729)72:3\ 453::AID-IJC13[3.0.CO;2-D. 25. Moot DT, Mobley HL, Chippendale GR, Lewison JF, Resau JH. Helicobacter pylori urease activity is toxic to human gastric epithelial cells. Infect Immun. 1990;58:1992–4. 26. Malmsten C, Kindahl H, Samulsson B, Levy-Toledano S, Tobelem G, Caen JP. Thromboxane synthesis and the platelet release reaction in Bernard-Soulier syndrome, thrombasthenia Glanzmann and Hermansky-Pudlak syndrome. Br J Haematol. 1977;35:511–20. doi:10.1111/j.1365-2141.1977.tb00617.x. 27. Wucherpfennig KW. Mechanisms for the induction of autoimmunity by infectious agents. J Clin Invest. 2001;108:1091–104.

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