Γενετικοί και μοριακοί παράγοντες συσχετίσεως λοιμώξεως από ελικοβακτηρίδιο του πυλωρού και γλαυκώματος

Abstract

Chronic open-angle glaucoma is a progressive optic neuropathy, which derives from a multitude of risk factors; it comprises mainly primary open-angle (POAG) and pseudoexfoliation (PXG) glaucoma. Approximately 2.5 million Americans 40 years of age or older suffer from POAG, and half of those affected may not even be aware that they have it. In early disease stages, glaucoma is asymptomatic but gradually it affects the visual field, leading to blindness if left untreated. The burden on the disease is disproportionately borne by persons of black African descent, and is globally, according to the World Health Organization, the third leading cause of blindness in order of frequency, after cataract and uncorrected refractive errors (near-sightedness, far-sightedness or astigmatism), affecting approximately 66 million people of whom at least 6.8 million are blind bilaterally.It would be desirable to be able to predict the glaucoma risk potential for an individual based on specific characteristics, traits, and habits of that individual. Unfortunately, our level of knowledge of glaucoma risk factors is inadequate in attaining this goal. While glaucoma constitutes a multifactorial disease, considerable evidence supports intraocular pressure (IOP) as an ocular risk factor for POAG, which remains, at present, the only risk factor that can be managed therapeutically. Apart from the increased IOP, other risk factors include older age, race (African), a family history of glaucoma, vascular factors such as hypertension, vasoconstriction, atheromatosis or acute low blood pressure, a risk factor for normal-tension glaucoma, and possibly infections. In 2000, Kountouras et al. first reported that H. pylori infection and glaucoma might be associated causatively, based on epidemiological data showing that both are diseases of older adults in the developed world. Moreover, chronic H. pylori infection may produce systemic disorders involving vascular tone resulting from the release of vasoactive and proinflammatory substances, such as ischemic heart disease, migraine, or Raynaud’s phenomenon, which represent manifestations of vascular dysregulation also present in the pathogenesis of glaucoma. Finally, H. pylori infection is associated, like glaucoma, with arteriosclerosis-induced increased platelet activation and aggregation.The existing published data pertaining to the prevalence of H. pylori infection in patients with different glaucoma types present geographic and ethnic similarities or diversities reflecting the geographic and ethnic variations in the prevalence of H. pylori infection and the different glaucoma types.More specifically, the Greek data show a high prevalence of H. pylori infection in Greek patients with POAG and XFG, H. pylori eradication was shown to benefit glaucoma progression, suggesting a possible causal link between the bacterium and glaucoma, and H. pylori-specific IgG antibody concentration was increased in the aqueous humor of POAG patients and correlated with the degree of vertical cupping, possibly indicating the severity of glaucomatous damage. These data indicate that H. pylori infection seems to be involved in the pathogenesis of glaucoma.By applying various relevant diagnostic methods for the detection of H. pylori infection, studies from Korea, China, India, Iran and Turkey published later, were in accordance with the Greek data, whereas studies from Canada, Italy and Israel, based on the serological diagnostic method failed to prove a relationship between H. pylori infection and glaucoma.However, it has been emphasized that although the serological test establishes the presence of H. pylori infection, it does not discriminate between current and old infections. Such a distinction is crucial because only current H. pylori infection induces humoral and cellular immune responses that, owing to the sharing of homologous epitopes (molecular mimicry), cross-react with components of nerves, thereby contributing and possibly perpetuating the apoptotic neural tissue damage, characterizing the neurodegenerative disorders, including the glaucomatous apoptotic neuropathy. Genetic background in conjunction with the mentioned H. pylori-induced humoral and cellular immune responses may contribute in the pathophysiology of glaucoma and other neurodegenerative diseases by inducing various relative mediators such as oxidative stress, inflammatory cytokines or oncogenes. Moreover, eradication of H. pylori infection might delay the development or progression of neurodegenerative diseases, including glaucoma, particularly at early disease stages.The aim of this study was: (1) to reconfirm in a new cohort of POAG patients the initial evidence that an increased prevalence of H. pylori infection is found in POAG patients, (2) to investigate the potential presence of H. pylori infection in incisional biopsy specimens obtained from trabeculum, iris, and conjunctiva of POAG patients during trabeculectomy surgery (in situ), (3) to test the initially shaped hypothesis that apoptosis may be a common pathogenetic mechanism of both diseases, by assessing oncogenes, oncosuppressive genes related to apoptosis or anti-apoptotic proteins, and cellular immune markers, (4) to investigate possible genetic factors associated with both diseases, by investigating the relationship between HLA antigens/alleles and HLA subtypes in patients with POAG and H. pylori infection, and (5) to investigate particularly the role of oxidative stress in the pathogenesis of these two diseases.For this purpose, this study focused on the following: a. Investigation of H. pylori infection prevalence (mainly using histologic diagnostic method with a few patients tested with urea breath test) in a new cohort of POAG patients compared with a control group.b. Histologic evaluation of the presence of H. pylori bacteria locally (trabeculum / iris / conjunctiva).c. Immunohistochemistry of gastric biopsy specimens for evaluation of:i. oncogene Ki-67ii. oncosuppressive gene p53, involved in apoptosisiii. anti-apoptotic gene Bcl-2iv. cellular indices of immune surveillance by measuring Σ-lymphocytes (TLs, UCHL-1) and B-lymphocytes (ΒLs, CD20).d. Quantitative measurement of serum interleukins (IL) -1β, -2, -6, -8, -10, interferon-γ, tumor necrosis factor-α and endothelin-1 by ELISA.e. Analysis of HLA subtypes A, B, C, DR and DQ by PCR techniques of our POAG patients compared with the bone marrow donors database of the Histocompatibility Department of Hippokration General Hospital of Thessaloniki, Greece.f. Assessment of oxidative stress.In the present study, a total of 51 consecutive patients, who underwent trabeculectomy for POAG not responsive to topical anti-glaucoma therapy, were included. Presence of H. pylori was established by histology, or urea breath test in 8 patients not eligible or who refused to undergo endoscopy. Thirty-five consecutive participants with mild iron-deficiency anemia who underwent upper gastrointestinal endoscopy and histologic examination for H. pylori presence served as controls. Moreover, in POAG patients we evaluated: (a) the expression of oncogenes p53, Ki-67, Bcl-2, and parameters of T-lymphocytes (TLs) (UCHL-1) and B-lymphocytes (BLs) (CD20) by immunohistochemistry; (b) serum IL -1β, -2, -6, -8, -10, interferon-γ, tumor necrosis factor-α and endothelin-1 by ELISA; (c) HLA subtypes A, B, C, DR and DQ by PCR techniques; and (d) reactive oxygen metabolites (ROMs) in capillary blood by spectrophotometry.Our results show that:Η. H. pylori infection was detected histologically or by urea breath test in a high rate of our POAG patients (84.3%), further reinforcing the results of our previous studies.ΗΗ. H. pylori bacteria were detected by histology in incisional biopsy specimens from the trabeculum and iris of 5 POAG patients.ΗΗΗ. UCHL-1 was positively expressed in all POAG patients with H. pylori infection and CD20 was positively expressed only in one POAG patient with H. pylori infection.IV. Ki-67, p53 and Bcl-2 were over-expressed in 19%, 25% and 37.5%, respectively, of the POAG patients with H. pylori infection.V. Cytokine levels in serum did not differ statistically between H. pylori positive and negative POAG patients.VΗ. HLA-A33 and HLA-B35 were statistically more frequent compared to a healthy population. High-resolution analysis showed that HLA-DQB1*0301 and HLA-DQA1*0505 were more frequent in this cohort of POAG patients.VII. ROMs were significantly more increased in H. pylori-positive than in H. pylori-negative POAG patients.These results show that H. pylori bacteria are detected histologically in biopsy specimens obtained from the trabeculum and iris of POAG patients that, together with the presence of the infection in the gastrointestinal tract, may induce humoral and cellular immune responses, possibly suggesting a role of various apoptotic or anti-apoptotic genes, mainly TLs, cytokines, oxidative stress and genetic factors in the pathophysiology of POAG.

Πρόσφατα δεδομένα μας δείχνουν αυξημένη συχνότητα της Helicobacter pylori λοιμώξεως σε Έλληνες ασθενείς με πρωτοπαθές γλαύκωμα ανοικτής γωνίας (POAG), η θεραπεία εκριζώσεως έχει θετική επίπτωση στην διετή πορεία του γλαυκώματος και ο αυξημένος τίτλος των anti-H. pylori IgG αντισωμάτων στο υδατοειδές υγρό γλαυκωματικών ασθενών δυνατόν να αντανακλά την βαρύτητα της γλαυκωματικής βλάβης. Σκοπός της διατριβής ήταν: (1) να επιβεβαιωθεί σε νέο δείγμα ασθενών η αρχική διαπίστωση ότι υφίσταται αυξημένη συχνότητα H. pylori λοιμώξεως σε ασθενείς με POAG, (2) να διερευνηθεί τυχόν εντόπιση του H. pylori σε οφθαλμικό ιστό (in situ), (3) να ελεγχθεί η υπόθεση ότι η απόπτωση δυνατόν να αποτελεί κοινό παθογενετικό μηχανισμό, εκτιμώντας ογκογονίδια, ογκοκατασταλτικά γονίδια ή αντι-αποπτωτικές πρωτεΐνες, και κυτταρικούς δείκτες ανοσιακής επιτηρήσεως, (4) να διερευνηθεί πιθανή γενετική επίπτωση στην εκδήλωση των δύο νόσων εκτιμώντας τη συσχέτιση των HLA αντιγόνων/αλληλίων στους ασθενείς με γλαύκωμα και H. pylori λοίμωξη, και (5) να διερευνηθεί ο ρόλος του οξειδωτικού στρες στην παθογένεια των δύο νόσων. Στην παρούσα μελέτη συμπεριελήφθησαν 51 ασθενείς που υπεβλήθησαν σε τραμπεκουλεκτομή εξαιτίας POAG που δεν απαντούσε σε τοπική αντι-γλαυκωματική αγωγή. Η παρουσία της H. pylori λοιμώξεως διαπιστώθηκε ιστολογικώς, ή με δοκιμασία ουρίας αναπνοής σε 8 ασθενείς που δεν κρίθηκαν ικανοί ή αρνήθηκαν να υποβληθούν σε ενδοσκόπηση ανωτέρου πεπτικού. Στην ομάδα των μαρτύρων συμπεριελήφθησαν 35 ασθενείς με ήπια σιδηροπενική αναιμία οι οποίοι επίσης υπεβλήθησαν σε ενδοσκόπηση ανωτέρου πεπτικού και ιστολογική εξέταση για παρουσία H. pylori λοιμώξεως. Επιπλέον, στους POAG ασθενείς, εκτιμήθηκαν: α) με ανοσοϊστοχημεία η έκφραση των ογκογονιδίων p53, Ki-67, Bcl-2 και παράμετροι των T-λεμφοκυττάρων (UCHL-1) (CD20) και B-λεμφοκυττάρων (CD20), β) οι ιντερλευκίνες -1β, -2, -6, -8, -10, η ιντερφερόνη-γ, ο tumor necrosis factor-α και η ενδοθηλίνη-1 του ορού με ELISA, γ) οι HLA υπότυποι με τεχνικές PCR και δ) οι δραστικοί μεταβολίτες οξυγόνου (ROMs) με σπεκτροφωτομετρία. Από την ανάλυση των ευρημάτων διαπιστώσαμε: Ι. Παρουσία H. pylori λοιμώξεως ιστολογικώς ή με δοκιμασία ουρίας αναπνοής σε μεγάλη αναλογία των ασθενών μας με POAG (84,3%). ΙΙ. Ιστολογική παρουσία H. pylori βακτηρίων σε 5 ασθενείς σε δείγματα βιοψιών από σκληροκερατοειδικό διηθητικό ηθμό (trabeculum) και ίριδα. ΙΙΙ. Θετική έκφραση UCHL-1 στο σύνολο των POAG ασθενών με H. pylori λοίμωξη και θετική έκφραση CD20 έκφραση μόνο σε έναν POAG ασθενή με H. pylori λοίμωξη. IV. Υπερέκφραση των Ki-67, p53 και Bcl-2 στο 19%, 25% και 37,5%, αντίστοιχα, των ασθενών με POAG και H. pylori λοίμωξη. V. Παρουσία κυτταροκινών ορού που δεν διέφεραν σημαντικά μεταξύ H. pylori θετικών και αρνητικών ασθενών. VΙ. Σημαντικά συχνότερη παρουσία υποτύπων HLA-A33 και HLA-B35 σε σύγκριση με υγιή πληθυσμό και συχνότερα αλλήλια HLA-DQB1*0301 και HLA-DQA1*0505 στους POAG ασθενείς της μελέτης. VIΙ. Σημαντική αύξηση των ROMs στους H. pylori-θετικούς έναντι των H. pylori-αρνητικών POAG ασθενών. Τα ευρήματα αυτά δείχνουν ότι H. pylori βακτήρια ανιχνεύονται ιστολογικώς σε δείγματα βιοψιών του διηθητικού ηθμού και ίριδας POAG ασθενών που σε συνδυασμό με την εντόπιση της λοιμώξεως στην γαστρεντερική οδό πιθανόν να επάγουν χυμικές και κυτταρικές ανοσιακές αποκρίσεις οι οποίες συμβάλλουν με ποικίλους μηχανισμούς στην παθοφυσιολογία του POAG όπου πιθανόν εμπλέκονται γονίδια αποπτωτικά ή αντιαποπτωτικά, κυρίως T-λεμφοκύτταρα, κυτταροκίνες, οξειδωτικό στρες και γενετικοί παράγοντες.