This is an author version of the contribution published on:
Questa è la versione dell’autore dell’opera:
[Minerva Biotec, 29 (3), 2017, 10.23736/S1120-4826.17.02220-0]
ovvero [Marco ASTEGIANO, Giovanni A. TOUSCOZ, Gian P. CAVIGLIA, Davide G.
RIBALDONE, Claudio DE ANGELIS, Sergio PEYRE, Antonina SMEDILE, Giorgio M.
SARACCO, Rinaldo PELLICANO, 29, ed. Minerva Medica, 2017, pagg.109-113]
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La versione definitiva è disponibile alla URL:
Non-organ specific autoimmunity in patients suffering from gastric ulcer
with and without Helicobacter pylori infection
Marco ASTEGIANO1, Giovanni Antonio TOUSCOZ1, Gian Paolo CAVIGLIA2, Davide Giuseppe RIBALDONE1, Claudio DE ANGELIS1, Sergio PEYRE3, Antonina SMEDILE1,2, Giorgio Maria SARACCO1,2, Rinaldo PELLICANO1
1Unit of Gastroenterology, Molinette Hospital, 10123 Turin, Italy
2Department of Medical Sciences, University of Turin, 10123 Turin, Italy 3U.O.A. Gastroenterology, Cuorgné Hospital, 10082 Cuorgné, Italy
Corresponding author: Rinaldo Pellicano, Unit of Gastroenterology, Molinette Hospital,
10123 Turin, Italy. Tel: +39 (0)11 6333918; Fax: +39 (0)11 6333623; e-mail:
rinaldo_pellican@hotmail.com
Conflict of interest statement: The authors certify that there is no conflict of interest
with any financial organization regarding the material discussed in the manuscript.
This work was not supported by grants.
Abstract
BACKGROUND: To evaluate the prevalence of non-organ specific autoantibodies (NOSAs) in patients with gastric ulcer (GU) associated or not with Helicobacter pylori (H. pylori) infection.
METHODS: We compared 43 patients (26M/17F, median age 63 [range 47-73] years)
suffering from GU and H. pylori infection to 39 subjects (19M/20F, median age 69 [range 52– 77] years) affected by GU caused by the assumption of non-steroidal anti-inflammatory drugs (NSAIDs). H. pylori infection was diagnosed by histology and 13C-urea breath test.
Autoimmunity was assessed by the presence of NOSAs in serum. All patients with H. pylori infection were treated with one or more antibiotic regimens to eradicate the bacterium. RESULTS: Antinuclear (ANA), anti-smooth muscle (ASMA) and anti-liver/kidney
microsomal-1 (LKM-1) antibodies were present respectively in 5 (11.6%), 1 (2.3%) and none out of 43 patients with GU and H. pylori infection. Considering the 39 patients with NSAIDs-related GU, ANA was present in 5 (12.8%), ASMA in 3 (7.7%) and anti-LKM-1 in none. The difference was not significant. All but one H. pylori infected subjects (97.7%) eradicated the bacterium with a multistep strategy; in none of them significant changes in terms of
prevalence or titer of NOSAs, after 6 months of follow-up were observed.
CONCLUSIONS: In patients with GU, H. pylori infection is not associated with an increased prevalence of NOSAs and the cure of the bacterium does not influence the NOSAs profile.
Key words: Autoantibodies - Autoimmunity - Helicobacter pylori - Gastric ulcer - Peptic
Helicobacter pylori (H. pylori) is a Gram-negative bacterium, infecting approximately 50% of the world’s population. The natural habitat of H. pylori is the antral mucosa of the stomach, where the micro-organism induces gastritis and peptic ulcer disease.1 Furthermore, to date the bacterium is recognized as a necessary, but insufficient, cause of gastric
adenocarcinoma and a strong association between H. pylori infection and gastric mucosa-associated lymphoid tissue (MALT) lymphomas has been proven by epidemiologic, biological, and molecular genetic studies.2, 3 In the last two decades several studies have reported on the link between chronic H. pylori infection and a variety of extra-gastric manifestations,4 based on a potential mechanism involving a low-grade inflammatory state, molecular mimicry patterns, and interference with absorption of nutrients.5, 6
The potential capacity of H. pylori role to trigger an autoimmune response in gastric mucosa has been attributed to bacterial lipopolysaccharide (LPS). This is supported by the evidence that the structure of LPS O-specific antigen is similar to LewisX and LewisY blood group determinants, which are also expressed in the normal glandular gastric epithelium.7 Hence, such molecular mimicry might account for some of the gastric autoantibodies induced by H. pylori infection. The same pathogenetic model have been proposed for various
autoimmune disorders, including systemic rheumatic diseases,8 thyroid diseases and idiopathic thrombocytopenic purpura reported in association with H. pylori.9, 10
We have previously reported that the seroprevalence of non-organ specific
autoantibodies (NOSAs) in a cohort of patients with duodenal ulcer (DU) is not increased in presence of H. pylori compared with uninfected patients.11 At difference of DU, gastric ulcer (GU) has a pathogenesis based on a weakness of parietal structure and is accompanied with a diffuse or a corpus-predominant atrophic gastritis, associated to a glandular destruction. This justify the decreased acid output showed in these patients. The role of H. pylori in the cascade of events leading to GU is crucial and is supposed to involve a disruption of the mucosal
defence, as a result of the intense inflammation.12 However, the exact mechanism remains unclear and the role of an autoimmunitary pathway, mediated by NOSAs, cannot be ruled out. For this reason, we have assessed the prevalence of NOSAs in patients suffering from GU comparing those infected by H. pylori versus the uninfected ones.
Materials and Methods
PatientsA total of 82 patients with GU were enrolled at the outpatient clinic of the
Gastroenterology Unit of Molinette Hospital and at the Gastroenterology Unit of Cuorgné Hospital. The presence of NOSAs was evaluated in 43 consecutive patients (26M and 17F, median age 63 [47–73] years) suffering from GU and H. pylori infection, and in 39
consecutive subjects (19M and 20F, median age 69 [52–77]) affected from GU caused by the assumption of non-steroidal anti-inflammatory drugs (NSAIDs) and without H. pylori
infection.
Patients previously treated for H. pylori, or with autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, infection by hepatitis B and C viruses, toxic or metabolic hepatitis, were excluded. In addition, patients with known autoimmune disorders were considered ineligible for the study.
All included patients gave their written informed consent prior to recruitment. The study protocol conformed to the principles of the Declaration of Helsinki.
Indirect immunofluorescence
Autoimmunity pattern was demonstrated by the presence of NOSAs in serum as recommended by International guidelines.13 A serum sample from each patient was tested to detect anti-nuclear (ANA), anti-smooth muscle (ASMA) and anti-liver/kidney microsomal-1
(LKM-1) antibodies. Sera diluted 1:40 in phosphate-buffered saline (PBS) were tested on cryostat sections of snap-frozen rat liver, kidney, and stomach. Fluorescein-conjugated antihuman immunoglobulin (DAKO FITC- Conjugated Rabbit Anti-Human IgA, IgG, IgM, Kappa, Lambda, Fluorescein Isothiocyanate Conjugate, DAKO, Glostrup, Denmark) was used as secondary antibody and diluted 1:50 to 1:100 in PBS, according to the manufacturer’s instruction. The immunofluorescence (IF) patterns were assessed under fluorescence
microscope (Eclipse 600, Nikon Corporation, Tokio, Japan). To better define the ANA pattern, all sera were also evaluated on commercially available HEp-2 cell lines (Diasorin, Saluggia, Italy) at 1:40 dilution. The presence of ASMA in serum samples was assessed using rat kidney and stomach as substrate. Anti-LKM-1 antibodies were detected by IF on rat liver and kidney sections; sera defined positive were subsequently tested by a competitive enzyme linked immunosorbent assay (ELISA) using reference antibody, as described elsewhere.11
Diagnosis of Helicobacter pylori infection
The search for the bacterium was performed by biopsy samples obtained during endoscopy (at least two in the gastric antrum and two in corpus/fundum) and, in case of negativity, the 13C urea breath test (UBT) was utilized. For the latter, the Helicobacter Test INFAI® (INFAI, Bochum, Germany) was used following the instruction of the manufacturer. The reported sensitivity was 97.9% and the specificity 98.5%. Two breath samples were obtained at baseline and thirty minutes after the sequential drink of 200 ml orange juice and a 75 mg dose of 13C-labeled urea in 50 ml of water. Samples were analyzed for 13C/12C ratio with a mass spectrometer (BreathMAT plus, Finnigan, Bremen, Germany). Results were expressed as excess 13CO2 excretion per mil, which represents 13C enrichment over and above the baseline sample: a value 4 delta per mil was considered positive. In case of confirmed infection, a commercial enzyme immunoassay (Helori-CTX® Eurospital, Trieste, Italy) was
used to detect serum IgG directed to the cytotoxin associated protein (CagA) of H. pylori, marker of more virulent strains of the bacterium. The assay sensitivity and specificity were 94% and 97%.
All patients with H. pylori infection underwent to a clarithromycin-based triple therapy.14 In non-responders, a metronidazole-based triple therapy (second step) and, eventually, a levofloxacin-based triple therapy (third step) were administered. The result of the antimicrobial treatment was confirmed by histology (when repetition of endoscopy was considered appropriate) and/or by 13C-UBT.
Statistical analysis
Continuous variables were reported as median (range) whereas categorical data as number and percentage. Data normality was checked by D’Agostino Pearson test. Difference between continuous variables was assessed by non-parametric Mann-Whitney test, while difference between categorical variables was evaluated by Fisher exact test. Contingency tables larger than 2 rows and 2 columns were tested by chi-squared test. Results were considered significant when p < 0.05. All statistical analysis was performed using MedCalc software version 12.7.0.0 (MedCalc, Ostend, Belgium).
Results
Patients demographic and clinical data are reported in Table I. No difference was observed regarding age, gender distribution, tobacco and alcohol consumption, and family history of GU, amongst groups. ANA, ASMA and LKM-1 antibodies were present,
respectively, in 5 (11.6%, all women), 1 (2.3%, woman) and none of the 43 patients with GU and H. pylori infection. Considering the 39 patients with NASIDs-related GU, ANA was present in 5 (12.8%, all but one women), ASMA in 3 (7.7%, one woman) and anti-LKM-1 in
none of the subjects. The difference was not significant (Table II). Moreover, no difference was found between the 2 groups regarding gender and autoantibodies-positivity interaction (p=0.209). Autoantibody titer was 1:40 in 5 patients with GU and H. pylori infection and 1:80 in the remaining case; in those with NASIDs-related GU, 5 out of 8 patients had a titer of 1:40 and in the remaining 3 the titer amounted to 1:80 (p=0.580).
As expected, among subjects with GU and H. pylori infection, 42 out of 43 (97.6 %) had antibodies against CagA. All patients with autoantibodies and H. pylori infection also had antibodies directed against the more cytotoxic strains of the bacterium.
All but one H. pylori infected subjects (97.6%) eradicated the bacterium with the above reported strategy; in none of them was observed significant changes with respect to prevalence or titer of NOSAs, after 6 months of follow-up.
Discussion
The issue of H. pylori infection on patients with autoimmune disorders and vice-versa should be carefully evaluated because it opens clinical and experimental questions. In general, a process of molecular mimicry may stimulate auto-reactivity in a genetically susceptible host, triggering a subsequent autoimmune response. H. pylori chronic infection is responsible of immune system activation and continuation of inflammatory response with ongoing cytokine production, infiltration of gastric mucosa by neutrophils and lymphocytes, finally leading to B cell response with antibodies production.15
H. pylori infection, in combination with environmental factors and autoimmunity, is strongly associated with atrophic gastritis development.16 In a study it has been reported that autoantibodies directed to parietal cells (APCA) were positive in 9.9% and 0% of H. pylori-positive and -negative subjects respectively.17 In addition, another study showed higher APCA levels in patients with severe atrophic lesions of the gastric mucosa in comparison with those
with mild atrophy, suggesting that APCA plays a relevant role in disease progression after H. pylori infection.18 It has been hypothesized a cross-reactivity between strong immunogenic surface proteins of H. pylori and human heat shock protein (Hsp).19 Thus, the bacterium would induce, in its host, antibodies against Hsp 60, which cross-react with the human Hsp60;20 this cross-reaction was reputed to be able to trigger an autoimmune reaction leading to atrophic gastritis and gastric cancer.21 Furthermore, immunization or infection of
experimental animals with H. pylori induces anti-H, K-ATPase autoantibodies due to molecular mimicry.22
The biological basis for the link between H. pylori and autoimmune disease is
represented by the abnormal imbalance in Th cell subsets during this infection. In particular, a relative increase of Th 1 cells,23 known to play a role in cell-mediated immunity, has been demonstrated, while Th 2 cells, a subset which is normally involved in the generation of a mucosal secretory immune response, does not seem to be stimulated.24
On the basis of these observations, it could be hypothesized that an antigenic similarity between H. pylori and host antigens is responsible for autoimmunity in some infected
subjects. However, no proof exists of the bacterium triggering production of autoantibodies of systemic relevance.
Conclusions
The results of our paper demonstrate that the prevalence of NOSAs, in patients with H. pylori infection is similar to that of controls. In addition, bacterial eradication did not modify the autoimmunitary pattern. In conclusion, in this study, the putative role of H. pylori in inducing organ-specific autoimmunity does not hold true for non-organ-specific
Conflict of interests
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TABLE I. Study population.
GU + H. pylori NSAIDs-related GU p value
Patients, n 43 39
Age (years), median (range) 63 (47-73) 69 (52-77) 0.088
Sex, M/F 26/17 19/20 0.375
Cigarettes smokers, n (%) 18 (41.8%) 14 (35.9%) 0.653 Alcohol drinkers, n (%) 21 (48.8%) 16 (41.0%) 0.512 Family history of GU, n (%) 5 (11.6%) 6 (15.4%) 0.749
Abbreviations: F, female; GU, gastric ulcer; H. pylori, Helicobacter pylori;M, male; NSAIDs, non-steroidal anti-inflammatory drug.
TABLE 2. Presence of autoantibodies in the population in study.
Antibodies GU + H. pylori NSAIDs-related GU p value* p value**
Patients, n 43 39
ANA positive, n (%) 5 (11.6%) 5 (12.8%) 1.000
ASMA positive, n (%) 1 (2.3%) 3 (7.7%) 0.342 0.512
LKM-1 positive, n (%) 0 0 /
*p values were calculated by Fisher exact test ** p value was calculated by chi-squared test
Abbreviations: ANA, antinuclear antibodies; ASMA, anti-smooth muscle antibodies; F, female; GU, gastric ulcer; H. pylori, Helicobacter pylori; LKM-1, anti-liver/kidney microsomal-1; M, male; NSAIDs, non-steroidal anti-inflammatory drug.
