Chapter 1. Introduction
1.2 Chronic infection and cognitive impairment
Chronic infection that is caused by some infectious agents, such as herpes simplex virus, Chlamydia pneumonia, latent toxoplasmosis and Helicobacter pylori (H. pylori) may induce neuroinflammation and exacerbate AD [11, 12]. Clinical and basic studies provide evidence that inflammation triggered by infectious agents is related to the pathogenesis of ischemic stroke, atherosclerosis, diabetes and dementia [13, 14].
Bacterial or viral invades vessel walls, provoke cytokine release, influence lipid
metabolism, contribute to other ways to vascular dysfunction. Based on human autopsy brains, Gramms and Ovase demonstrated that AD microvessels released significantly higher levels of interleukin (IL)-1beta, IL-6, and tumor necrosis factor α compared with non-AD microvessels [15]. In addition, levels of microvessel-associated monocyte chemoattractant protein and IL-1beta were high in AD-derived microvessels, but not detectable in non-AD microvessels. Cerebral microcirculation contributes inflammatory mediators to the microenvironment of the AD brain and may be involved in the
pathogenesis of neuronal injury [16]. Neurotoxic events may be triggered by central nervous system (CNS) immunocompetent cells (microglia and astrocytes) at CNS or by infiltrating peripheral monocytes and leukocytes (T-cells and B-cells). These cells produced some factors that may directly contribute to neurotoxicity; these factors include antibodies, complement proteins, inflammatory cytokines, chemokines, glutamate, kynurenine metabolites, and reactive oxygen and nitrogen species. These inflammatory factors of CNS diseases have been associated to the immune response to damage in the affected brain area [15]. Moreover, the neuroimmune axis also played a role in the etiology of neurodegenerative diseases involving slow, chronic and
progressive neuropathology. In other words, the pathological change of neuronal loss found in AD and brain viral infections may be induced directly by immune activation
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[17].
1.3 Chronic H. pylori infection and cognitive impairment
Among pathogens associated with cognitive impairment, H. pylori is especially known for its chronic course and its capability to be eradicated effectively. H. pylori, a spiral-shaped gram-negative bacterium, colonizes in the stomach and may lead to chronic gastritis, peptic ulcers and gastric cancer. Most of the time, the infection is acquired in early childhood and causes lifelong gastric inflammation [18]. Epidemiological studies revealed that the prevalence of H. pylori infection in Taiwan is around 43.9-54.4% [19, 20].
Previous studies exploring the association between H. pylori infection and dementia have shown inconsistent findings. A meta-analysis including 7 studies found a significant association between H. pylori infection and dementia with a pooled odds ratio of 1.71 [21]. Moreover, two interventional studies and one large study based on claimed database found that successful H. pylori eradication may decelerate cognitive decline in AD and reduce mortality risk [22-24].However, previous studies differed in study design, study population, sample size, outcome assessment (e.g., cognition assessed by different tools), specific subtypes of dementia, and the type of assays used for evaluating the H. pylori infection. More details of the effect of H. pylori infection on cognitive function are summarized in Table 1-1. In addition, basic research also showed inconsistent findings.
For example, two animal studies found that a 7-day course of intraperitoneal injections of H. pylori filtrates led to amyloid deposition [25] and tau phosphorylation [26] in the brains of wild-type rats. However, one recent study did not confirm these findings [27].
Postulated mechanism. It is postulated that H. pylori infection may play a role in neuronal damage, the subsequent poor cognition and AD. First, chronic atrophic gastritis
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due to H. pylori infection has known to reduce the absorption of both vitamin B12 and folic acid, followed by secondary hyperhomocysteinemia [28-31], which leads to vascular dementia and AD via precipitation of endothelial damage and neurodegeneration [32]. H.
pylori also induces irregular humoral and cellular immune responses via sharing the
homologous epitopes, which cross-react with nerves and then lead to the apoptotic neural tissue damage observed in neurodegenerative diseases [33-35]. Third, systematic inflammatory responses that are induced by H. pylori infection release various cytokines (IL-1, IL-6, IL-8, IL-10, IL-12, tumor necrosis factor α, and interferon γ), acute-phase proteins [fibrinogen and C-reactive protein (CRP)], eicosanoids (leukotrienes and prostaglandins), defensins, and platelet aggregation, which may lead to both apoptosis of neural tissue and vascular thrombosis [33, 36]. In addition, a recent rat study demonstrated that an intraperitoneal injection of H. pylori filtrate led to increasing Aβ42 levels and interrupted synaptic function, which then affected spatial learning and memory [25]. The postulated mechanism based on previous studies for the relation between H.
pylori infection and cognitive impairment was shown in Figure 1-1.
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1.4 Chronic serum inflammatory markers, one carbon metabolism and cognitive impairment
Previous investigations into the interactions between H. pylori infection and serum inflammatory markers on cognitive performance are limited. Berrett et. al. reported H.
pylori interacted with CRP and the inflammatory index (standardizing the concentration
counts for fibrinogen, ferritin, and CRP and summing them to create a standardized composite score) were associated with worse processing speed in adults aged 20–59 [37].
However, inflammatory markers (e.g., fibrinogen, ferritin and CRP) or folate did not mediate the relationship between H. pylori and cognitive function [37]. Beydoun et al.
found that H. pylori infection was associated with markers reflecting the iron status (e.g., serum ferritin and transferrin saturation), which then altered factors involved in one carbon metabolism (e.g., folate, vitamin B 12 and homocysteine), and subsequently affected the levels of some antioxidant status (e.g., vitamins A and E) [38]. Recently, Berrett et. al. further demonstrated that H. pylori seropositivity interacted with 5-methyltetrahydrofolate concentration to predict poor performance on the digit symbol coding task [39].
Inflammation seems to enhance the detrimental effect of high levels of homocysteine on cognitive functioning. The joint effect of hyperhomocysteinemia and inflammation may promote the development and progression of atherosclerotic plaques, which is an important risk factor for dementia [40, 41]. Homocysteine is synthesized
from methionine and it can either transformed back to methionine or convert
into cysteine with the aid of the B-group vitamins [39]. Elevated serum homocysteine level could induce venous thrombosis, promote the synthesis of several
pro-inflammatory cytokines in the arterial wall and has been associated with increased inflammation [42, 43]. Previous studies showed that higher homocysteine at baseline
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was associated with prolonged lower cognitive functioning and a faster rate of cognitive decline [44, 45]. The detrimental effect of higher homocysteine on the performance of immediate recall was strongest in persons with a high level of IL-6. Moreover, in the highest tertile of CRP, higher homocysteine was associated with poor memory retention [45]. In summary, hyperhomocysteinemia may contribute to immune activation and inflammation; the decline in cognitive function and neurogenesis throughout the progression of AD may be affected by autoimmune mechanisms reacting in the periphery and in the brain [46].
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1.5 Research gaps and study aims Research gaps
Previous studies focused on global cognition or a single cognitive domain, which are difficult to clarify the association between H. pylori infection and domain-specific cognition. In addition, most previous studies failed to incorporate the information of some important factors ( e.g., APOE ε4 status, inflammatory markers, and depressive symptoms) [47].
Study aims
This study aimed to assess the association between global and domain-specific cognition in relation to chronic H. pylori infection among the elderly. We also collected information on the aforementioned factors and performed a stratified analysis by age groups (65-74 vs. ≥75 years old), APOE ε4, sex, inflammatory markers and factors involved in one carbon metabolism to examine the aforementioned association. The framework of this research was shown in Figure 1-2. In addition, we aim to perform mediation and moderation analysis to clarify if inflammation and one carbon
metabolism play a role in H. pylori related cognitive impairment.
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Chapter 2. Materials and Methods
2.1 Study design and population Study population
Participants aged 65 years or older were recruited from the annual elderly health checkup (EHC) at the National Taiwan University Hospital (NTUH) from 2011 to 2013.
Participants with the following conditions were not recruited into this study (1) major organ dysfunction (heart failure, decompensated liver cirrhosis, and end stage renal disease, etc.), (2) advanced stage of cancer, and (3) unable to complete the cognitive tests by himself or herself. A total of 605 participants were recruited. Among them, participants with the following conditions or diseases were further excluded (n=18): use of medication(s) for AD treatment (n=2), history of stroke (n=9), and a lack of data on H.
pylori infection (n=1) and cognitive tests (trail making test A and B, n=6). After exclusion,
a total of 587 participants were included for statistical analysis. The flow chart of the study population was shown in Figure 2-1.
Ethics statement
The study protocol was approved by the Research Ethics Committee (REC) of NTUH.
Written informed consent was obtained from each study participant before the administration of questionnaires, performance of clinical assessments, and collection of biological specimens at NTUH. This study complies with the World Medical Association Declaration of Helsinki.
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2.2 Cognitive assessment
Cognitive assessment
Both global and domain-specific cognition were assessed using various neuropsychological tests. Global cognitive function was assessed by the Montreal Cognitive Assessment-Taiwanese version (MoCA-T) [48]. The MoCA-T score ranges from 0 to 30 and is composed of five cognitive domains (i.e., memory, visuospatial, attention, language, and orientation). Compared with Mini-Mental State Examination (MMSE), the MoCA-T is a more sensitive instrument than the MMSE to screen AD patients from non-dementia cases [49-51].
For domain-specific cognition, the Wechsler Memory Scale-Third Edition (WMS-III) was used to assess logical memory (logical recall I & II, and logical memory-thematic I & II) and attention (digit span-forward & backward) [52]. Verbal fluency tests included tasks such as naming fish, vegetables and fruit within 1 minute; sum of the three above tests was recorded as the total score of verbal fluency. Trail making tests (A and B) were used to assess executive function. Eleven domain-specific cognitive variables were obtained from these neuropsychological tests for a subsequent exploratory factor analysis (EFA). Each cognitive score was used to estimate a z-score based on the mean and standard deviation (SD) of the study population. Global cognitive impairment for Taiwanese was defined as a MoCA-T score < 24, mild cognitive impairment (MCI) was defined as a MoCA-T score of 22 or 23, and normal cognition was defined as a MoCA-T score ≥ 24. This cutoff score was validated in a Taiwanese population with a sensitivity of 92% and a specificity of 78% [48]. For evaluation of the visuospatial function of subjects, the visuospatial domain which including connecting, square drawing and clock drawing tests) was independently extracted from the MoCA-T as one of the major
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outcome of this study.
Neuropsychological tests
Logical memory. The original WMS was published by Wechsler in 1945 and then revised in 1987, 1997 and 2009. WMS is one of the most frequently used instrument in clinical settings to assess memory of people aged 16 to 90 years-old. The WMS consists of seven subsets, which were designed to measure different aspects of memory, including logical memory, learning, working memory and attention. The seven subsets were personal and current information, orientation, mental control, logical memory, digit span, visual reproduction and associate learning. The logical memory subset is composed of two stories that were verbally presented by the examiner to the study participants. One story is about a woman and the other story is about a man. Immediately after each story is presented, the study participant is asked to repeat what they remember without giving any clues which is to assess logical memory-immediate recall. Each story contains 25 units. The memory scoring system credits one point for each recall of one specific unit in the story (for example, the character’s name). Another scoring criteria considers the recall of general topics (theme) which has a total of 7 points for the first story and 8 points for the second story. For example, the theme scoring system grants a point if the participant recalls that the story is about a woman, even if the character’s name is not remembered.
Thirty minutes later, the study participant is asked to recall the story again which is used to assess logical memory-delayed recall. For both immediate and delayed recall, all correct items are summed giving a maximum score of 25 for each story. The logical memory domain in our study was composed of four scores: logical memory-recall I & II, and logical memory-thematic I & II.
Executive function. Executive function was measured by trail making test A and B [53]. Trail making test A requires an individual to draw lines sequentially connecting 25
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encircled numbers distributed on a sheet of paper. Task requirements are similar for trail making test B except the person must alternatively link the numbers to the letters (e.g., 1 to 甲 to 2 to 乙 to 3 to 丙, etc.). The score of each part represents the amount of time required to complete the task with a ceiling value of 300 seconds. Unlike the other cognitive tests performed in this study, a longer time spent on trail making A test or B indicates worse cognitive function.
Verbal fluency. Verbal fluency is assessed by three verbal fluency tests [54]. Verbal fluency test assesses the language function and may also involve in the performance of executive function [55]. The participant is asked to list all of the fruits he/she can think of in 60 seconds. Similarly, this test was repeated by asking the participants to list all kinds of the fish and vegetables he/she can think of in 60 seconds. One point was granted for each item of fruit, fish or vegetables provided in 60 seconds.
Attention. Digit span-forward and backward components in the WMS-III were used to assess attention [56]. Participants are presented with a series of digits (e.g., 8-2-4) and must immediately repeat them back. If they do this successfully, a longer list (e.g., 9-2-4-1) are given to them. The length of longest list a participant can remember is the score of the participant’s digit span. While the participant is asked to enter the digits in the given order in the forward digit-span task, the participant needs to reverse the order of numbers in the backward digit-span task. Totally 8 tests were given in digit span-forward and there are two subtests in each test. Totally 7 tests were given in digit span-backward and there are two subtests in each test.
The measurement methods and scoring range/criteria of neuropsychological tests were summarized as Table 2-1.
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Exploratory factor analysis for identification of cognitive factor
With the use of EFA, cognitive factors were identified based on the 11 domain-specific variables mentioned above. All cognitive factors were subsequently rotated by an orthogonal transformation (varimax rotation) to achieve better clustering. Four cognitive domains with an eigenvalue > 1 demonstrated a clear turning point on the scree plot, and neuropsychological tests with an absolute value of a factor loading ≥ 0.5 were selected for naming a specific cognitive factor. The factor score for each cognitive factor was estimated by summing all of the weighted neuropsychological test scores by their factor loadings [57]. Each cognitive factor was labeled according to the main psychological test within that factor.
Cognitive impairment was defined as the lowest tertile (T1) of the z-score for domain-specific cognitive variables and cognitive factors; normal cognition was defined as the higher tertiles (T2+T3).
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2.3 Covariates
A self-report questionnaire was administered by trained interviewers to collect the information on demography, comorbidity and lifestyle. Each participant underwent a physical examination, which included body weight, height and blood pressure. Body mass index (BMI) was estimated by dividing body weight (kg) by the square of body height (m2). Hypertension was defined as a self-reported history of hypertension, or the use of anti-hypertensives. Diabetes mellitus (DM) was defined as a fasting plasma glucose level
≥ 126 mg/dl, a self-reported history of DM, or undergoing current treatment for DM.
Physical activity was assessed by a short version of the International Physical Activity Questionnaire (IPAQ) [58]. Depressive symptoms were assessed by the Center for Epidemiological Studies Depression scale (CESD) [59], and a CESD score ≥ 16 points, self-reported depression, or the use of antidepressants all indicated the existence of depressive symptoms.
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2.4 Laboratory assay
A fasting venous blood sample was collected from each participant in the morning after an 8-hour fasting period.
Serum H. pylori IgG level
H. pylori infection was assessed by a serology test of IgG using a commercial enzyme
immunoassay (EIA) (IMMULITE 2000, Siemens, Germany) with a reported sensitivity and specificity of > 90%. All assays were performed and interpreted by a single laboratory investigator who was unaware of the sample status. The antibody titer ranged from 0.4 to 8.0 U/mL with a clinical cutoff point of 1.1 U/mL. In addition, the titer of H.
pylori serology does not provide a method for predicting the presence of ulcer in patients with H. pylori infection, but may imply the severity of histological changes [60].
Other serum biomarkers
The plasma levels of CRP and homocysteine were determined by TBA-200FR (Toshiba Medical Systems, Japan); vitamin B12 and folate levels were determined by an ABBOTT Architect i2000 analyzer (Abbott Park, Illinois, U.S.A.). Serum creatinine, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), triglyceride (TG) and fasting glucose were included in the package of the elderly health checkup program. The presence of IL-6 was tested by enzyme linked immunosorbent assay (ELISA) and was determined by a Tecan Infinite M200 multimode reader (Tecan, Männedorf, Switzerland).
Genotyping assay
Genomic DNA was extracted from buffy coat using a QuickGene-Mini 80 system
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(Fujifilm, Tokyo, Japan) and then stored in a -80°C freezer. The APOE ε4 status was determined by two single nucleotide polymorphisms (SNPs) s42938 and rs7412 [61]
using TagMan Genomic Assays by the ABI7900HT fast real-time PCR system (Applied Biosystems Inc., California, U.S.A.). Quality control samples were obtained from 5% of the internal samples provided by the manufacturers and then duplicated and genotyped together with all of the other samples, and the concordance rate was 100%. The Hardy-Weinberg equilibrium (HWE) test was performed among controls to examine possible genotyping error and selection bias for each SNP.
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2.5 Statistical analyses
Participants were quartiled according to their serum H. pylori IgG level. For descriptive analyses, analysis of variance (continuous variables) and chi-square tests (categorical variables) were used to compare different exposure variables across the quartiles of serum H. pylori IgG level. Multivariable linear regression models were used to examine the
change of global and domain-specific cognition scores per one unit increase of serum IgG level (i.e., the regression coefficient β). Multivariable logistic regression models were performed to estimate the odds ratio (OR) and 95% confidence interval (CI) for the association between serum H. pylori IgG level and cognitive impairment (i.e., MoCA-T score < 24 for global cognition; the lowest tertile (T1) of Z-score for specific cognitive domain test; or the lowest tertile (T1) of factor score of each cognitive factor). The covariates that were adjusted in the model included factors with biological importance for cognition (e.g., age, sex, years of education, and APOE ε4 status) and variables that were selected by the stepwise model selection (SLENTRY=0.15, SLSTAY=0.15). Stratified analyses were performed by important covariates [age groups (65-74 vs. ≥75), sex, and APOE ε4 status] for the association between the quartiles of serum H. pylori IgG and
cognitive impairment. A trend test (Ptrend) was performed for testing the relationship between the four quartiles of serum H. pylori IgG level and cognitive tests. We also evaluated how those covariates modified the association between H. pylori IgG level and the risk of cognitive impairment by comparing the model with main effect terms and interaction terms to the model with main effect terms only (Pinteraction < 0.10 indicated the existence of an interaction). A risk estimate (i.e., OR) reaches statistical significance when its corresponding 95% CI does not include 1. Statistical analyses were performed using SAS software version 9.3 (SAS Institute Inc., Cary, NC, U.S.A.) and all of the tests were
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two-sided.
Statistical power
EpiInfoTM 7 (Centers for Disease Control and Prevention, Atlanta, U.S.A.) was used to estimate the statistical power. According to previous studies, given the ratio of uninfected participants to infected participants as 1.2 and the effect size was 0.10 and 0.18 for the ratio of global cognitive impairment in uninfected and infected participants respectively, a sample size of 583 gave a statistical power of 80 % at a significance level of 0.05. This study included 587 participants, which provided sufficient statistical power to assess the association between H. pylori infection and cognitive impairment.
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2.6 Mediation and moderation analyses
To further understand the relationship between H. pylori and cognitive impairment, we performed mediation and moderation analyses to test if folic acid, vitamin B12, homocysteine, CRP or IL-6 play a role in the aforementioned association.
Mediation analyses
Mediation analyses were firstly described by Baron & Kenny in 1986 to explore the mechanism through which the causal variable affects the outcome. The mediator has been called an intervening or process variable [62]. The mediation model was shown in Figure 2-2 and included the following four steps to establish mediation:
Mediation analyses were firstly described by Baron & Kenny in 1986 to explore the mechanism through which the causal variable affects the outcome. The mediator has been called an intervening or process variable [62]. The mediation model was shown in Figure 2-2 and included the following four steps to establish mediation: