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The H+, K+-ATPase, present in the parietal cells of the gastric oxyntic mucosa, is a gastric proton pump that functions to maintain an acidic environment within the stomach by transporting H+ and K+ ions against their concentration gradients using energy derived from the hydrolysis of ATP. H+, K+-ATPases are P-type ATPases existing as heterodimers: the alpha-subunit, coded by the ATP4A gene on human chromosome 19q13.1, contains the catalytic site and mediates ion transport; the beta-subunit, coded by the ATP4B gene on human chromosome 13q34, acts to stabilize the alpha-subunit and is essential for enzyme function.1, 2, 3 Circulating autoantibodies targeting this H+/K+-ATPase proton pump of the gastric parietal cell are considered diagnostic markers in patients with autoimmune gastritis and pernicious anemia,4, 5, 6, 7 and currently, the diagnosis of autoimmune gastritis relies on the demonstration of these autoantibodies.8 Autoimmune gastritis is a chronic disorder with a prevalence of ~2% in the general population and up to 10% in patients with other autoimmune diseases, such as type I diabetes or autoimmune thyroid disease.9, 10, 11, 12, 13, 14 Atrophic body gastritis (AGB) is the pathological lesion of autoimmune gastritis, characterized by the disappearance of oxyntic mucosa leading to reduced or absent secretion of gastric acid.15, 16, 17, 18, 19 From a pathogenetic point of view, atrophic body gastritis is a highly complex condition, which may be considered as consisting of three groups: (i) classical autoimmune H. pylori-negative atrophic body gastritis with a spared antrum; (ii) multifocal atrophic body gastritis involving the antral mucosa with active H. pylori infection and often negative for parietal cell autoantibodies, and (iii) a third group with overlapping features: body atrophy with a normal or inflamed antrum, active or past H. pylori infection and positivity to parietal cell autoantibodies.8 H. pylori-related atrophic body gastritis and autoimmune gastritis are linked to increased risk for gastric cancer and type 1 gastric carcinoids8, 20 and the need of endoscopic surveillance in these patients has been argued both in Europe and in the USA,21, 22 thus making screening and timely diagnosis of this condition an important issue. Autoantibodies against parietal cells are currently used as a serological marker to screen patients with other autoimmune disorders for autoimmune gastritis.7, 8 In patients with type I diabetes, autoimmune thyroid disease, and vitiligo, positivity to parietal cells autoantibodies have been reported in up to 25%,10, 11 32%,13, 14, 23 and 8%24 of patients, respectively. Despite their current use as serological markers, the diagnostic utility of these circulating autoantibodies remains largely unknown and studies on their diagnostic accuracy in patients with atrophic body gastritis are lacking. Positivity to autoantibodies against parietal cells has been shown in 7.8–19.5% of the general population.25, 26 Previous studies mainly employed enzyme immunoassays (EIA) or indirect immunofluorescence to detect autoantibodies against parietal cells that are reactive to either ATPase subunit (4A and/or 4B) of the gastric pump.7, 27 Some studies have argued that the H+, K+-ATPase 4A subunit (ATP4A) may be the major antigen,28, 29 but only few studies focusing on antibodies directed towards the H+, K+-ATPase 4B subunit have been performed so far.30 The aim of this study was thus to assess the presence of autoantibodies against ATP4A and ATP4B subunits of parietal cells H+, K+-ATPase in patients with atrophic body gastritis and controls. The presence of atrophic body gastritis was defined on the basis of histological confirmation of gastric corporal mucosal atrophy, fasting hypergastrinemia, and low pepsinogen I levels.5, 12 All patients underwent gastroscopy with standardized biopsy sampling from the antrum (n=3) and body (n=3) mucosa for conventional histopathological examination. The degree of gastritis was assessed according to the updated Sydney System.31 Atrophy of the gastric body mucosa was defined as focal or complete oxyntic gland loss and/or their replacement by metaplastic pyloric or intestinal glands.5, 12 H. pylori immunoglobulin G antibodies were determined using a commercial ELISA kit (GAP test IgG, Bio-Rad, Milan, Italy). ABG patients were defined as having active H. pylori infection when both histology and ELISA serology were positive.5, 12, 32 The presence of pernicious anemia was defined as the presence of a macrocytic anemia (hemoglobin concentration <14 g/dl for males and <12 g/dl for females, mean corpuscolar volume≥100 fl) associated with low levels of serum cobalamin (normal values 190–950 pg/ml) and response to vitamin B12 treatment.33 All cases and controls were of Caucasian origin. None of the patients or controls included in the study were on treatment with anti-secretory drugs (proton pump inhibitors and/or H2 antagonists). The study was performed in a blinded fashion, so that the case–control status of samples as well as patients’ characteristics were unknown to the investigators performing the serological analyses. The study was approved by the local Ethical Committees, and patients and controls provided signed informed consent. All LIPS analyses were performed at the IRCCS San Raffaele Scientific Institute Division for Genetics and Cellular Biology, Milan, Italy, and the researchers who performed the LIPS measurements (VL, CB and IM) were blinded to the clinical data and to the diagnoses of cases and controls. In 83 (79.8%) cases and 185 (90.2) controls, autoantibodies against parietal cells were assessed by using a commercial EIA kit (QUANTA LiteTM GPA, INOVA Diagnostics, San Diego, USA, cut-off <20 units). As shown in Figure 1a,b, the ATP4A and ATP4B autoantibody titers were significantly higher in cases (ATP4A: median 3003 AU, range 54–47,429 AU; ATP4B: median 2,762 AU, range 29–19,796 AU) compared with controls (ATP4A: median 1 AU, range 1–2,440 AU; ATP4B: median 2 AU, range 0.2–1,301 AU; P<0.0001 for both assays). For the ATP4A assay, the area under the ROC curve was 0.98 (95% CI 0.965–0.996) with a significance level (area=0.5) of P<0.0001. For the ATP4B assay, the area under the ROC curve was 0.99 (95% CI 0.979–1.000) with a significance level (area=0.5) of P<0001. As shown in the scatter diagram of correlation between ATP4A and ATP4B autoantibody titers (Figure 2), a statistically significant correlation between the two assays was shown (Spearman's coefficient of rank correlation (rho)=0.895. 95% CI 0.870–0.915, P<0.0001) indicating a parallel reactivity against both, the ATP4A and the ATPB antigens. Based on ROC curve analysis, a threshold for positivity for each assay was selected corresponding to the highest value at which maximal sensitivity was achieved. For the ATP4A assay, this corresponded to 52 AU and yielded a sensitivity of 100% (104/104) and a specificity of 89% (23/205). For the ATP4B assay, this corresponded to 28 AU and yielded a sensitivity of 100% (104/104) and a specificity of 90% (20/205). We then adopted as a classifier for atrophic gastritis or control status the simultaneous positivity in both assays. Based on this decision algorithm, the sensitivity in the atrophic gastritis patients was stable at 100% (104/104) while specificity in control increased to 95% (10/205; Table 2). As shown in Table 2, the specificity and positive predictive values of EIA, 92 and 81%, respectively, were similar to those of ATP4A and ATP4B LIPS assays, while the 72% sensitivity was significantly lower compared with the 100% sensitivity values of the LIPS assays (P<0.0001). Ten (43.5%) of the 23 controls with positivity to ATP4A and eight (40%) of the 20 controls with positivity to ATP4B had a positive anti-H. pylori IgG test, consistent with a potential role of H. pylori infection in triggering reactivity against ATP4A and/or ATP4B in a subgroup of control subjects. The ATP4A and ATP4B autoantibody titers of the “false positive” controls were significantly lower as compared with the “true positive” atrophic body gastritis patients (ATP4A: median 299 AU (range 51–2,440 AU) vs. median 2,949 UA (range 54–47,428 AU), P<0.0001; ATP4B: median 367 UA (range 218–1,301 AU) vs. median 2,761 (range 29–19,796 AU), P<0.0001). As shown in Figure 3 and Table 4, patients with atrophic body gastritis having a spared antral mucosa (the typical histological feature of autoimmune gastritis) had a significantly higher ATP4A and ATP4B autoantibody titer compared to those having an inflamed or atrophic antral mucosa (more typically linked to H. pylori-correlated atrophic gastritis). The ATP4B autoantibody titer, but not the ATP4A autoantibody titer, was also significantly higher in patients with concomitant autoimmune thyroid disease and in those without active H. pylori infection (Table 4), suggesting a closer relationship of this autoantibody to gastric autoimmunity. This is supported by the analysis of the 5 atrophic body gastritis patients who were negative for ATP4B autoantibodies: they were aged between 21 and 56 years and all were female, but only one had a spared antrum, none had severe atrophy, intestinal metaplasia or pernicious anemia, only two had associated autoimmune thyroid disease and all five were H. pylori positive (4 with active infection at histology and 1 with positive serology). The LIPS assays for the parietal cell K+/H+ ATPase described herein show a high diagnostic performance for atrophic body gastritis, with 100% sensitivity for both, the ATP4A and the ATP4B assays. According to our results, IgG autoantibodies against the ATP4A and ATP4B antigens are virtually always present in patients with atrophic body gastritis and thus they seem to represent reliable serological biomarkers of oxyntic mucosa damage tout court suggesting a close relationship of oxyntic mucosa atrophy and the expression of these autoantibodies. Biomarker tests have been defined as clinically useful when they result in improved measurable clinical outcomes of value to clinical decision-making compared with routine management without use of the test.41 Autoantibodies against parietal cells are often used to screen for autoimmune gastritis in patients with other autoimmune diseases:7, 8 in patients with type I diabetes, autoimmune thyroid disease, and vitiligo, positivity to parietal cells autoantibodies up to 25%,10, 11 32%13, 14, 23 and 8%,24 respectively, has been reported. Atrophic body gastritis is a condition linked to an increased risk of gastric neoplasia.8, 19, 42, 43 To better address invasive and cost-intensive gastroscopy (currently essential for diagnosis of atrophic body gastritis), noninvasive screening tools such as serum pepsinogen I and gastrin have been proposed.34, 44 According to a recent meta-analysis, screening for atrophic gastritis by serum pepsinogens has 69% sensitivity at 88% specificity and an 88% AUC.31 Another screening tool which combines gastrin 17, pepsinogens I and II and H. pylori antibody levels showed 50% sensitivity at 80% specificity for atrophic gastritis.45 According to the results of the present study, the assessment of IgG autoantibodies against ATP4A and/or ATP4B may be proposed as biomarker not only for autoimmune gastritis, but also for other forms of atrophic body gastritis, and positive patients should be advised to undergo gastroscopy with biopsies to establish diagnosis of atrophic body gastritis and to rule out neoplastic complications of this condition. This is the first study assessing the diagnostic potential of autoantibodies to ATP4A and ATP4B both separately and in combination using LIPS in subjects with atrophic body gastritis, thus no previous data for comparison of our results are available. However it should be noted that Burbelo et al.30 reported that 2/90 (2.2%) of control subjects in the 2010 Diabetes Autoantibody Standardization Program showed ATP4B positivity using a related LIPS assay, yielding a higher specificity compared to the 10% “false positive” rate of the current study. Most previous studies employed EIA or indirect immunofluorescence to detect autoantibodies against parietal cells, which do not discriminate reactivity to specific ATPase subunits of the gastric pump.7, 27 Typically EIA is the preferred method, as it shows high concordance among laboratories and is ~30% more sensitive than immunofluorescence.7, 27 Recently, a novel radioimmunoprecipitation assay was reported for the ATP4A subunit,28 which shows similar performance characteristics to the LIPS assay described above (VL, HWD & JMW; data presented at the 13th Meeting of the Immunology of Diabetes Society, Lorne, Australia, December 2013). Compared with EIA, radioimmunoprecipitation assays are usually a more accurate and precise method for autoantibody assessment, but have the disadvantage of requiring special laboratory conditions and instrumentation.7 The high diagnostic accuracy for atrophic gastritis of ATP4A and ATP4B autoantibodies observed in this study might be explained by at least two reasons. First, sensitive LIPS assays were employed for assessment of these autoantibodies. LIPS, is a technique that offers several advantages over current alternatives. For example, solid phase assays such as Western blotting, EIA and protein arrays, often have a narrow dynamic range for measuring autoantibodies and may show sub-optimal detection of conformational epitopes, even after optimization for background noise. In contrast, liquid phase assays such as radioimmunoprecipitation and LIPS readily detect autoantibodies directed against both conformational and linear epitopes, offering high sensitivity and specificity for many autoimmune diseases. In addition, since protein targets are genetically fused to luciferase, there is no need to purify the antigens, and no use of radioactivity. Together, these advantages suggest that LIPS is an ideal platform for the assessment of autoantibodies in a wide range of diseases including atrophic body gastritis.38, 46, 47 The results in the present study showed that LIPS assay outperformed traditional EIA yielding a significantly higher sensitivity and resetting to zero the proportion of (“false”) negative atrophic gastritis patients to autoantibodies against H+,K+-ATPase antigens. Second, all of the included cases with atrophic body gastritis had a histologically proven presence of gastric mucosa atrophy which was severe in 42.3%, with associated intestinal metaplasia in 59.6%, and spared antrum (completely healthy antral mucosa) in 44.2%. Thus, the LIPS assays against ATP4A and ATP4B tested a well characterized specific target population with histological diagnosis of atrophic damage and a high likelihood of positivity to parietal cell autoantibodies. Therefore, the diagnostic accuracy of this assay as a screening tool needs to be validated in a population of subjects undergoing biopsy with suspected (but not confirmed) disease. The LIPS assays described above show high specificity; only 11.2 and 9.7% controls tested positive for ATP4A and ATP4B, respectively, a figure which was similar to that observed with commercial EIA (7.6%). H. pylori infection has been associated with anti-parietal cell autoantibodies, with positivity reported in up to 20.7% of H. pylori positive patients.25, 26 In our study, ~40% of the ATP4A or ATP4B positive controls had a positive H. pylori serology, possibly explaining the reactivity against the ATPase as a consequence of the infection in this subgroup, as previously shown and linked to antigen mimicry.48, 49 In the remaining ATP4A or ATP4B positive controls who tested negative for H. pylori, the “false positivity” may be explained by an unspecific autoreactivity as described for anti-mitochondrial, anti-nuclear, and anti-smooth muscle autoantibodies.50, 51 In a cross-sectional study performed in Spain, besides H. pylori infection, other factors associated with autoantibodies against parietal cells were female gender, insulin resistance, menopause, and high serum levels of soluble CD40 ligand.26 A recent study assessing the presence of ATP4A autoantibodies in diabetic children also failed to show an association between H. pylori serology and positivity to ATP4A autoantibodies,35 suggesting that factors other than H. pylori may also lead to autoreactivity against the parietal cellsATPase. In our study, female gender between cases and controls was not different, but controls were slightly younger than cases (49 vs. 56 years, P<0.05). It has been reported that positivity to anti-parietal cell antibodies increases with age.27 Thus, we cannot exclude the possibility that the specificity observed for ATP4A and ATP4B autoantibodies in this study might overestimate that in the general population. Some differences emerged between the autoreactivity against the ATP4A and ATP4B subunits. A spared antrum, the classical histological feature linked to autoimmune gastritis, was associated with higher autoantibody titers against ATP4A and ATP4B, compared with multifocal atrophic gastritis. Other features more closely linked to autoimmune gastritis, such as an absence of active H. pylori infection and concomitant autoimmune thyroid disease, were associated with higher autoantibody titers against ATP4B only. The ATP4A subunit is believed to be the major antigen,28, 29 while the ATP4B subunit is supposed to stabilize the alpha-subunit and to perform essential enzyme function.1, 2, 3 These results suggest that the reactivity against the different ATPase subunits might differ according to different clinical features of atrophic body gastritis patients, possibly offering some insights regarding the pathogenetic role of these autoantibodies to be clarified in future studies. In conclusion, positivity to autoantibodies against both the ATP4A and ATP4B subunits, is closely associated with atrophic body gastritis. Both, the ATP4A and the ATP4B assay had the highest sensitivity, at the cost of diagnostic accuracy (89 and 90% specificity), outperforming traditional EIA. Once validated, these LIPS assays should be valuable screening tools for detecting biomarkers of damaged atrophic oxyntic mucosa.  Guarantor of the article: Bruno Annibale, MD. Specific author contributions: study design: Edith Lahner, Bruno Annibale, and Vito Lampasona; development of LIPS assays: Cristina Brigatti, Ilaria Marzinotto, Emanuele Bosi, Lorenzo Piemonti, Howard W Davidson, Janet M Wenzlau, and Vito Lampasona; LIPS assays: Ilaria Marzinotto and Vito Lampasona; data analysis: Edith Lahner, Giulia Scalese, Marilia Carabotti, and Bruno Annibale; first draft of the manuscript: Edith Lahner; and all authors contributed to its completion. All authors approved the final draft submitted. Financial support: B.A. acknowledge support from University Sapienza 2013–2014. H.W.D. and J.M.W. acknowledge support from NIH grant R01 DK052068 (to H.W.D.). C.B., I.M., L.P., E.B. and V.L. acknowledge their work was carried out within the framework of the “Ivascomar project, Cluster Tecnologico Nazionale Scienze della Vita ALISEI, Italian Ministry of Research”. Potential competing interests: None.

clinical and translational gastroenterology

Luminescent Immunoprecipitation System (LIPS) for Detection of Autoantibodies Against ATP4A and ATP4B Subunits of Gastric Proton Pump H+,K+-ATPase in Atrophic Body Gastritis Patients