Local and Systemic Immune and Inflammatory Responses to Helicobacter pylori Strains

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1 CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, Dec. 2005, p Vol. 12, No X/05/$ doi: /cdli Copyright 2005, American Society for Microbiology. All Rights Reserved. Local and Systemic Immune and Inflammatory Responses to Helicobacter pylori Strains Niranjan Bhat, 1 James Gaensbauer, 1 Richard M. Peek, 2,3 Karen Bloch, 1 Kyi-Toe Tham, 2,4 Martin J. Blaser, 5,6 and Guillermo Perez-Perez 5 * Division of Infectious Diseases, Vanderbilt University School of Medicine, Nashville, Tennessee 1 ; Veterans Affairs Medical Center, Nashville, Tennessee 2 ; Division of Gastroenterology, Vanderbilt University School of Medicine, Nashville, Tennessee 3 ; Department of Pathology, Vanderbilt University School of Medicine, Nashville, Tennessee 4 ; Departments of Medicine and Microbiology, New York University School of Medicine, New York, New York 5 ; and Veterans Affairs Medical Center, New York, New York 6 Received 19 July 2005/Returned for modification 28 July 2005/Accepted 4 October 2005 Colonization with Helicobacter pylori eventuates in varied clinical outcomes, which relate to both bacterial and host factors. Here we examine the relationships between caga status, serum and gastric juice antibody responses, and gastric inflammation in dyspeptic patients. Serum, gastric juice, and gastric biopsy specimens were obtained from 89 patients undergoing endoscopy. H. pylori colonization and caga status were determined by histology, culture, and PCR methods, and acute inflammation and chronic inflammation in the gastric mucosa were scored by a single pathologist. Serum and gastric juice antibodies to H. pylori whole-cell and CagA antigens were determined by enzyme-linked immunosorbent assay. Relationships between variables were sequentially analyzed using univariate and multivariate statistical methods. Of the 89 subjects, 62 were colonized by H. pylori. By univariate analyses, levels of serum immunoglobulin G (IgG) and IgA and gastric juice IgA antibodies against whole-cell and CagA antigens each were significantly higher in the H. pyloripositive group than in the H. pylori-negative group (P < 0.001). H. pylori and CagA seropositivities were both significantly associated with enhanced inflammation in gastric antrum and body (P < 0.02). The presence of gastric juice antibodies to H. pylori antigens was associated with more severe gastric inflammation. However, in multivariate analyses, only the presence of serum antibodies against CagA and, to a lesser extent, whole-cell antigens remained significantly associated with acute and chronic inflammation in antrum and body (P < 0.05). Thus, serum antibody response to CagA correlates with severity of gastric inflammation. Furthermore, given the relationships demonstrated by multivariate analysis, determination of gastric juice antibodies may provide a better representation of serum, rather than secretory, immune response. Helicobacter pylori is a persistent gastric colonizer of approximately half of the human population (46). Carriage of H. pylori is usually asymptomatic but is associated with an increased risk of gastric and duodenal ulceration, atrophic gastritis, and adenocarcinoma of the distal stomach in 10% of colonized individuals (5, 19, 26, 32, 58). The enhanced risk of these diseases is due to the ability of H. pylori to induce chronic inflammatory responses in the gastric mucosa (5). Since illness develops in only a fraction of carriers, it is important to identify host factors and H. pylori characteristics that affect the risk of developing disease. The production of an immunodominant 120- to 140-kDa protein encoded by the cytotoxin-associated gene A (caga) by some strains of H. pylori is one potential virulence factor that alters clinical manifestation. caga is present in approximately 60% of H. pylori strains in the United States (10, 51). The presence of caga and the serologic response to its product (the CagA protein) are markers for the presence of the cag pathogenicity island (24, 50), a 37- to 40-kb genomic region that functions to interact with the gastric epithelium (7, 22, 36, 52). Evidence indicates that persons who carry caga H. pylori strains develop a more pronounced inflammatory response (13, 42, 47) and have an increased risk of developing peptic ulceration and non-cardia gastric adenocarcinoma, compared to persons carrying caga-negative strains (6, 13). Analysis of these relationships has relied predominantly on the presence or absence of specific serum antibodies to the CagA protein (6, 12, 25, 40). Several investigations, however, have demonstrated a strong relationship between gastric mucosal inflammation or ulceration and gastric juice antibodies directed against H. pylori antigens (13, 23, 55), as well as to culture and PCR analysis of H. pylori obtained directly from gastric juice (1). These studies suggest that specific gastric juice antibody responses (mainly secretory immunoglobulin A [IgA]) to CagA may serve as biomarkers for pathological outcome. The goals of this study were to examine whether the caga status of H. pylori strains in dyspeptic patients or host differences in serum and gastric juice antibody responses to both H. pylori whole-cell (WC) and CagA antigens are related to gastric histopathologic findings. * Corresponding author. Mailing address: Division of Infectious Diseases, 6027W VAMC, New York University School of Medicine, 423 East 23rd St., New York, NY Phone: (212) Fax: (212) perezg02@med.nyu.edu. N.B. and J.G. contributed equally to this study. MATERIALS AND METHODS Study population. Study subjects were patients at the Nashville Veterans Affairs Medical Center who underwent gastroduodenal endoscopy for dyspeptic symptoms, as previously described (17). Patients were excluded if they were receiving steroids or other immunomodulating drugs, were abusing alcohol or 1393

2 1394 BHAT ET AL. CLIN. DIAGN. LAB. IMMUNOL. illicit drugs, or had taken antimicrobial agents within the prior 2 weeks. Patients currently using nonsteroidal anti-inflammatory drugs were included in the study population. All subjects gave informed consent for study participation. Of the original 130 subjects, adequate quantities of serum and gastric juice, complete histologic characterization of biopsies, and microbiologic characterization of the H. pylori isolates were available from 89 patients. There were no significant differences in patient diagnoses or demographics between these 89 studied patients and the 41 patients excluded from the study. Endoscopic procedures, quantitative H. pylori culture, and histologic analysis were performed as previously described (1). Upper endoscopy was performed in the morning after an overnight fast which began at midnight. All gastric juice samples were obtained early during the endoscopy after entering the stomach. No extra information on gastric juice secretion was available. During endoscopy, gastric mucosal biopsies were obtained from the gastric corpus and the greater curvature of the gastric antrum. From each site, two gastric biopsy specimens were obtained for quantitative culture and two for histologic evaluation. For 83 (93%) of the 89 patients, all biopsies were obtained: for six patients, biopsy samples were available from a subset of gastric sites. Serum samples were also collected from each patient on the day of the endoscopic procedure. Biopsy specimen preparation and analysis. Gastric biopsy specimens were immediately transferred to individual preweighed sterile microcentrifuge tubes containing 50 l of saline at 4 C and processed within 1 h. Each biopsy specimen was homogenized and resuspended in 250 l of sterile saline. For quantitative cultures, serial 10-fold dilutions in saline were inoculated on Trypticase soy agar with 5% sheep blood (BBL, Cockeysville, MD) and incubated under microaerobic conditions (CampyPak Plus; BBL) at 37 C for 4 days. H. pylori was identified based on positive urease, catalase, and oxidase tests and by typical appearance on Gram s stain as described previously (17). Quantitative bacterial culture. We estimated the number of H. pylori present in a gastric biopsy using the methods previously described (25). Due to the intensive nature of serial dilutions for quantitative bacteriology (1), analysis was performed on only 40 of the 62 patients ultimately found to be carriers of H. pylori. RNA extraction. From each biopsy yielding H. pylori, mrna was extracted, and PCR analysis was performed for detection of caga status, as previously described (41). A lower limit of detection of caga positivity corresponding to 2 CFU was obtained with this assay, and results were analyzed by a blinded observer (41). Histological assessment. Histology was evaluated using microscopic examination by a single experienced pathologist (K.T.) unaware of the patients clinical diagnoses or the H. pylori strain characteristics. Adjacent 4- m paraffin sections were stained with hematoxylin-eosin and with modified Giemsa stain. Bacterial density was also quantified in the Giemsa sections by counting H. pylori organisms on the mucosal surface and in the foveolae and dividing by the number of high-power fields included. Areas of intestinal metaplasia were not colonized and thus were excluded from this analysis. Histologic features were graded using the hematoxylin-eosin sections, as described previously (1, 41). The features scored were infiltration of neutrophils, lymphocytes, and plasma cells; epithelial degeneration (irregular luminal cell borders, nuclear pyknosis, and cytoplasmic eosinophilia); mucus depletion; and epithelial erosion. Each feature was graded in both biopsies from each site (antrum or corpus), and a mean score for both chronic and acute inflammation from each side was determined. Serum antibody analysis. Anti-H. pylori IgG antibody levels in serum were determined by use of an enzyme-linked immunosorbent assay (ELISA) that has a sensitivity and specificity of 95% and 96%, respectively, as described previously (16, 45). The antigen used in this assay consists of a preparation of sonicated whole cells from five H. pylori isolates. An optical density (OD) ratio was calculated for each sample based on the mean optical densities at 405 nm of two assays per sample and the mean of two standardized positive control samples from the same ELISA plate. An OD ratio greater than or equal to 1.0 was defined as evidence of the presence of a H. pylori response, as described previously (6, 39). Excellent reproducibility was found using this assay, with a variance of duplicate test results of less than 5%. Serologic determination of CagA status was assessed by use of an ELISA to detect the presence of serum IgG directed against ORV220, a truncated recombinant CagA protein, as described previously (6). An absorbance index below 0.35 in this assay was considered negative, while a value above 0.35 was considered positive (25). This ELISA was validated to maximum sensitivity and specificity using sera from persons known by other methods to be colonized by caga or caga-negative H. pylori strains. Gastric juice antibody analysis. Anti-H. pylori and anti-caga IgA antibody levels in gastric juice were also determined by ELISA using the same antigens described above, with the following modifications. Gastric juice samples were stored at 70 C for a period between 1 and 5 years before they were tested. TABLE 1. Demographic and clinical characteristics of 89 patients undergoing endoscopy, according to H. pylori status a Parameter Negative Gastric juice samples were collected, diluted 1:1 in Tris-Trasylol (aprotinin, protease inhibitor) neutralization buffer (ph 7.6), and immediately frozen to 70 C. Prior to assay, samples were further diluted 1:4 in Dulbecco s phosphatebuffered saline with 2.5% bovine serum albumin. Samples were added to antigencoated wells, incubated for 1 h at 37 C, washed three times in dilution buffer, and then bound with extravidin-labeled alkaline phosphatase (Sigma) for 1 h. The presence of bound antibodies was detected by reaction with p-nitrophenol and measurement of absorbance at 540 nm. Statistical analysis. For the purposes of univariate analysis, comparisons between groups of different serum and gastric juice antibody statuses were done using two-tailed heteroscedastic Student s t tests. To eliminate the statistical effects of confounding variables, multivariate analysis was performed. The inflammation scores for patients with pathological confirmation of H. pylori gastric colonization were converted into dichotomous variables, stratified by gastric site and acuteness of inflammation. Acute inflammation was graded as either present (a score of 1 to 3) or absent (a score of 0). As nearly all patients had some degree of chronic inflammation, this variable was divided into a high level (a score of 2 to 3) or a low level (a score of 0 to 1) of inflammation. Serum whole-cell IgG and IgA antibody levels were normally distributed and thus were converted into quintiles. Levels of serum IgG antibody to CagA were categorized as absent ( 0.35) or present ( 0.35). Gastric juice antibody levels were dichotomized into undetectable (0) and detectable ( 0) groups. Four models were constructed to analyze the relationship between antibody levels and acute, chronic, antral, and fundal gastric inflammation. The exact binomial method was used to calculate 95% confidence intervals for proportions. The Chi-square test or Fisher s exact test was used to compare the proportion of patients with high or detectable antibody levels and gastric inflammation. All variables with a P value of 0.35 by univariate analysis were entered into a multivariate logistic regression model. Variables with a P value of 0.05 were considered statistically significant and retained in the final model. RESULTS H. pylori status Positive No. of subjects P value Mean SD age (yr) b % Caucasian % Male % with diagnosis of c : Peptic ulcer disease d Duodenal ulcer a H. pylori positivity was determined by culture from biopsy, expression of H. pylori genes by PCR analysis, or direct microscopic visualization on biopsy tissue. b P value from independent two-tailed t test. c Clinical diagnosis at time of endoscopy. Peptic ulcer disease includes subjects diagnosed with gastric ulcer, duodenal ulcer, or duodenal erosions. d P value from chi-square analysis. Demographic characteristics of the study population. Demographic data for the 89 subjects are presented in Table 1. The population consisted largely of male Caucasians, with an average age of 55 years. Diagnoses made during endoscopy included esophagitis, non-ulcer dyspepsia, duodenal erosions, duodenal ulcer, and gastric ulcer. Stratification of the population based on H. pylori status revealed differences in demographic characteristics for the two groups. As expected, the 62 H. pylori-positive patients tended to be older and had a higher frequency of peptic ulcer disease (Table 1). The 27 H. pylori-

3 VOL. 12, 2005 IMMUNE RESPONSES TO H. PYLORI 1395 Downloaded from FIG. 1. Comparative antibody responses to H. pylori and CagA antigens in patients undergoing gastric biopsy. (Panel I) Serum IgG antibody responses to H. pylori whole-cell antigen. (Panel II) Serum IgG responses to CagA antigen. (Panel III) Gastric juice IgA responses to whole-cell antigen. (Panel IV) Gastric juice IgA responses to CagA antigen. Group A represents H. pylori-negative patients (n 27) and H. pylori-positive patients (n 62). Group B represents CagA PCR-negative patients (n 23) and CagA PCR-positive patients (n 39). Group C represents patients CagA negative by serology (n 30) and CagA positive by serology (n 32). Group D represents patients CagA negative by PCR and serology (n 19) and CagA positive by PCR and serology (n 28). negative subjects were more likely to have a diagnosis of esophagitis or non-ulcer dyspepsia. H. pylori-specific antibody responses in serum and gastric juice. To characterize the range of host responses to H. pylori in our study population, specific antibodies against H. pylori WC antigen and CagA antigen were quantified in serum and gastric juice samples. As expected, the H. pylori group produced significantly higher mean antibody responses compared to the H. pylori-negative group in all assays of serum and gastric juice (P 0.05 in each case) (Fig. 1, group A, all panels). H. pylori subjects were then stratified by caga status, as determined by PCR, serology, or both (Fig. 1, groups B, C, and D). Between the caga and caga-negative groups, there was no difference in mean serum IgG or gastric juice IgA antibody levels against WC antigens, using either PCR or serologic criteria to define caga status, as expected. In contrast, by both diagnostic criteria, there were significantly higher levels of serum IgG antibodies to the CagA antigen among subjects carrying caga strains (P 0.001). Gastric juice IgA antibodies to CagA antigen were significantly higher only in subjects positive for CagA by the serum antibody criterion. To more stringently assess these relationships, subjects who were determined to be caga by both PCR and serologic criteria were compared to those who were negative in both categories. This comparison again showed significantly higher levels of anti-caga serum IgG antibodies in the patients colonized by caga strains (P 0.001), while serum and gastric juice antibody responses to the WC antigens showed no difference, as expected (Fig. 1, groups B and D, panels I, II, and III). The gastric juice IgA antibody response to CagA antigen, however, was no longer significantly different (Fig. 1, groups B and D, panel IV). These data indicate that serum and gastric juice antibodies to H. pylori WC antigens and gastric juice IgA antibodies to CagA are not affected by the caga status of the colonizing strain. The serum IgA response to whole-cell antigen was identical to the response in IgG (data not shown). Secretory IgA may be unstable during multiple freeze/thaw cycles, but, our results showed that, for the H. pylori-positive patients, there was no decline in secretory IgA response. Furthermore, we did not observe any correlation either direct or on November 18, 2018 by guest

4 1396 BHAT ET AL. CLIN. DIAGN. LAB. IMMUNOL. TABLE 2. Analysis of severity of gastric inflammation and quantitative culture among patient groups defined by H. pylori and CagA status Category c (no. of subjects) Antrum Mean inflammation score a Body Chronic Acute Combined Chronic Acute Combined No. of subjects d Quantitative culture b Mean logarithm of CFU H. pylori Negative NA e (27) Positive (61) P value f CagA PCR Negative (22) Positive (39) P value f CagA IgG g Negative (30) Positive (31) P value CagA PCR and IgG Negative (19) Positive (27) P value f a Mean inflammation scores determined as described in Materials and Methods. b Quantitative culture represents mean logarithm of CFU per milligram (wet weight) of biopsy tissue. c Categories as defined in the legend to Fig. 1. d Number of subjects for which culture data are available. e NA, culture data not applicable in H. pylori-negative group. f All P values in Table 2 are from independent two-tailed t tests. g CagA status as determined by an anti-caga serum IgG ELISA OD ratio to positive control of inverse between the time of storage and secretory IgA levels in the gastric juices tested. Although it is possible that storage in the freezer may affect secretory IgA, our current data do not indicate that this effect can explain the negative results observed. Relationship between H. pylori status and gastric mucosal inflammation. We next sought to assess the relationship between H. pylori status and intensity of gastric mucosal inflammation. Gastric antral and body mucosal biopsy specimens from 88 of the 89 patients were scored for acute and chronic inflammation; a combined inflammation score was also calculated (Table 2). Subjects colonized with H. pylori exhibited significantly higher mean antral and corpus inflammation scores in all categories, compared to noncolonized subjects (P in all cases). Comparison of mean severity of inflammation between patients carrying caga and caga-negative strains showed that essentially all scores were significantly higher in the former group (P 0.05), regardless of the definition used. Quantitation of colonizing bacteria, however, was not significantly different between the groups, regardless of the criteria used for determining caga positivity (Table 2), indicating that the ability to induce more intense gastric damage is not due to increased colonization density. Relationship of immune response to gastric mucosal inflammation. To determine which antibody markers may be associated with gastric inflammation, we performed both univariate and multivariate analyses, using a threshold to define the presence of an antibody response in colonized patients. For each assay, antibody producers were defined as subjects with an OD ratio greater than the mean OD ratio plus 2 standard deviations found in the noncolonized group of patients. Using this definition, the presence of serum IgA antibodies to H. pylori WC antigen was not significantly associated with increased inflammation scores of any type (Table 3), nor was the presence of a serum IgA response to CagA antigen significantly associated with inflammation (data not shown). In contrast, univariate analysis demonstrated that the presence of a serum anti-caga IgG response was associated with increased inflammation in both the antrum and corpus, regardless of chronicity (P 0.05) (Table 3). While no statistically significant association was demonstrated between inflammation and a serum

5 VOL. 12, 2005 IMMUNE RESPONSES TO H. PYLORI 1397 TABLE 3. Analysis of severity of gastric inflammation and quantitative cultures among 61 H. pylori-positive patients grouped by presence of specific serum and gastric juice antibodies Antibody class and H. pylori antigen recognized (no. of subjects) Mean inflammation score a Antrum Body Chronic Acute Combined Chronic Acute Combined Quantitative culture b No. of subjects c Mean logarithm of CFU Serum antibodies d IgA WO Nonproducers (39) IgA WC producers (22) P value e IgG CagA Nonproducers (22) Producers (39) P value e Gastric juice IgA antibodies WC Nonproducers (34) Producers (27) P value e CagA nonproducers (42) (19) a Mean inflammation scores determined as described in Materials and Methods. b Quantitative culture represents mean logarithm of CFU per milligram (wet weight) of biopsy tissue. c Number of subjects for which culture data are available. d Antibody nonproducers represent negative subjects with mean ELISA OD ratio of mean 2 standard deviations of the OD ratio from the H. pylori-negative group of subjects (n 27); antibody producers represent positive subjects with an OD ratio equal to or greater than the mean 2 standard deviations. e All P values in Table 3 are from independent two-tailed t tests. IgG response against WC antigen, most categories did suggest a trend in this direction (data not shown). Patients who produced gastric juice IgA antibodies to H. pylori WC antigens had significantly higher inflammation scores in gastric body biopsies (P 0.05 for each analysis) but not in antrum biopsies (Table 3). Similar comparisons of gastric juice anti-caga IgA antibody production showed significant differences in the presence of acute and combined inflammation in gastric body biopsies. Gastric juice antibody levels showed no significant relationship with quantitative cultures (Table 3). Multivariate analysis was then performed to identify independent predictors of gastric pathology. Among the variables studied, including patient demographics and serum and gastric juice antibodies levels, only serum antibody levels were significantly associated with increased levels of inflammation (Table 4). Serum IgG antibody to CagA was a significant predictor for increased severity of inflammation in each of the four models TABLE 4. Risk factors that correlated with increased gastric inflammation in multivariate analyses a Site/type of inflammation b Variable c Adjusted OR (95% CI) d P value Total antral Serum IgG to CagA 5.16 ( ) 0.01 Total body Serum IgA to WC antigens 1.61 ( ) 0.04 Serum IgG to CagA 4.78 ( ) 0.01 Total acute Serum IgG to WC antigens 1.72 ( ) 0.03 Serum IgG to CagA 7.13 ( ) Total chronic Serum IgG to CagA 9.53 ( ) 0.01 a Multivariate analyses included the following variables: age, race/ethnicity, levels of serum IgG and IgA to WC antigen, serum IgG to CagA, and gastric juice IgA to WC and CagA antigens. b Total inflammation in region of stomach (acute and chronic combined) or total inflammation by type (antrum and body combined). c Serum IgG to CagA (positive or negative) based on a preestablished OD threshold ( 0.35); results for serum IgG to WC antigens were stratified into quintiles. For each significant variable, prediction of pathology increased with higher levels of antibody. d OR, odds ratio; CI, confidence interval.

6 1398 BHAT ET AL. CLIN. DIAGN. LAB. IMMUNOL. studied (antral, body, acute, and chronic). Additionally, levels of serum antibodies to WC antigens, while not significant in univariate analyses, were significantly associated with total body and total acute inflammation, though to a much lower extent than were antibodies to CagA. Multivariate analysis revealed no independently significant correlations between gastric juice antibodies against either antigen and gastric inflammation. Relationship of antibody response, gastric inflammation, and quantitative culture to disease status. Within the group of 62 patients colonized with H. pylori, we also sought to determine if the clinical diagnosis of peptic ulcer disease (n 23) was associated with differences in antibody levels, inflammatory responses, or bacterial numbers, compared with those in patients without ulcerative disease (n 39). Patients with peptic ulceration had significantly lower acute inflammation scores in the gastric corpus (mean, ) than did the patients without ulcer disease (mean, ) (P 0.04). In addition, patients with peptic ulcer disease had lower levels of gastric juice IgA antibodies to H. pylori WC antigens (mean OD, ) than did patients without peptic ulcer disease (mean OD, ) (P 0.03). Quantitative bacterial culture and other serological and histological findings were not related to ulcer disease (data not shown). DISCUSSION In this study, we have shown that subjects colonized with H. pylori have serum and gastric juice antibody levels to WC and CagA antigens and subjects colonized with H. pylori caga strains had increased levels of serum IgG antibodies to CagA antigen, a response associated with more severe gastric inflammation, as demonstrated by multivariate analysis. In contrast, gastric juice antibody levels showed little independent relationship to inflammation or severity of disease. Our study was conducted using a homogeneous population, composed primarily of symptomatic male Caucasian veterans. While this distribution limits confounding demographic variables and improves the consistency of patient selection, endoscopic methods, histologic scoring, and sample preparation, it limits our ability to generalize to larger populations. The use of only symptomatic patients also limits our ability to understand the effects of H. pylori colonization on inflammation and antibody formation in asymptomatic persons, a distinction that may be particularly relevant to gastric juice antibodies. However, that these patients received a wide variety of clinical diagnoses demonstrates that symptoms were not necessarily correlated with specific clinical or histologic disease. Finally, while the sensitivity and specificity of the ELISA for gastric juice antibodies used in this study have not been previously characterized, gastric juice antibody production against H. pylori WC and CagA antigens in H. pylori-positive individuals was significantly correlated with serum antibody production, measured using a well-established assay (6, 45). An important strength of this study is that we performed a multidimensional analysis, including demographic and clinical factors, H. pylori strain characteristics, serum and gastric juice humoral responses, tissue responses to the organism, and H. pylori enumeration, based on quantitative culture rather than histological estimation. Examination of these data in multivariate analysis extends previous univariate analyses, including our own (8, 12, 14, 15, 40, 42), improving confidence in our understanding of the underlying phenomena. A wide body of evidence suggests that the presence of caga strains, or at least the presence of serum antibodies to the CagA antigen, is an important predictor of clinical outcome in Western populations (6, 13). This is not surprising, since cag strains inject the CagA protein into gastric epithelial cells (22, 36). In Asian populations, in which most H. pylori-positive persons carry caga strains, such relationships cannot be shown (31, 37) or, if present, are less clear cut (21, 48, 59). An important question that must be addressed is to define the gold standard for cag positivity. Culture-based methods are traditionally the gold standard for assessing microbial presence, but in using culture to assess caga status, several problems have emerged. First, humans may simultaneously carry both cag and cag-negative strains, and if only a single colony is used as the basis to determine cag presence, this may represent a false-negative result (18, 20, 49, 53). Second, even among persons known to carry cag strains, there may be a spontaneous loss of caga and other parts of the cag island (53, 57). Third, sequence heterogeneity and technical limitations may compromise PCR methodologies (54). Thus, mucosal and serum antibody analyses have an important advantage over culture-based methods for detecting cag positivity, since they are, in effect, global tests that sample the entire stomach. Host responses to H. pylori antigens vary widely (2), reflecting the highly variable interactions with these microbes (3, 5), and depending on the threshold used, serologies also may be falsely negative. Within our study, we found that four persons who were PCR negative for caga were positive by serology. Conversely, of 39 patients known by PCR to carry a cag strain, 12 did not meet the serologic criterion. Thus, to maximize accuracy, we used gold standard groups who were positive in both PCR and serologic assays or negative in both. Using this relatively stringent classification, the results in the univariate analyses were nevertheless nearly the same as when only PCR or the serologic criterion was used. Carriage of a caga strain was associated with increased acute and chronic cellular responses in both gastric antrum and body, in comparison to carriage of a caga-negative strain. For the first time, using multivariate analysis, we show that the presence of serum IgG antibodies to CagA is strongly associated with each of four interrelated measures of inflammation, classified as affecting the body or antrum (regardless of histological type) and as acute or chronic (regardless of anatomic site). The effects are consistent and large, with adjusted odds ratios ranging from 4.8 to 9.5. These results are compatible with the hypothesis that colonization with an H. pylori population consisting predominantly of caga strains induces a substantially different host response than does a predominantly caga-negative population (4). It has become increasingly clear that such differences also may be clinically relevant for the risk of development of such diseases as duodenal ulceration, noncardia gastric cancer (6, 10, 13, 34, 35), as well as Barrett s esophagus and its sequelae (9, 30, 43, 56), in Western populations. In addition to the importance of caga positivity on clinical outcome, these data indicate a smaller but independent association between serum IgG antibodies to WC antigens and

7 VOL. 12, 2005 IMMUNE RESPONSES TO H. PYLORI 1399 total body and total acute inflammation (adjusted odds ratio, 1.6 to 1.7). Observing a direct association of serum IgG antibodies to H. pylori WC antigens and the risk of development of noncardia gastric cancer and peptic ulceration, we earlier hypothesized that the increased antibody levels may reflect enhanced inflammation (33, 44). The current results support that hypothesis; however, they do not permit discrimination as to whether the higher serum antibodies may contribute to enhanced inflammation, as an immunoregulatory phenomenon, or merely reflect the extent of inflammation. While the effect of increased antibody levels is much smaller in magnitude than that associated with caga positivity, the height of these levels may have future clinical relevance in assessing individual patient risk. In this study, the level of gastric juice IgA antibodies to either the H. pylori WC or CagA antigen was significantly associated with acute inflammation of the gastric body in univariate analysis (Table 3), as previously observed (11, 13, 42). However, the lack of an association in the multivariate analysis suggests that this phenomenon is secondary to the underlying association of acute body inflammation with serum antibody levels. As a result, assessment of gastric juice antibody levels may not accurately represent secretory immune responses but merely reflect serum responses; we find no added utility as markers for enhanced tissue damage. A report from Thailand confirmed the poor sensitivity of gastric juice IgA to assess H. pylori status (27). Another potential explanation is that secretory IgA may be unstable when stored in the freezer. However, our results did not show a correlation between time of storage and secretory IgA levels in the gastric juice samples of the H. pylori-positive patients. Thus, although freezer storage could partially affect the secretory IgA levels, it is not sufficient to completely explain the negative results reported here. Quantitative culture results were not associated with inflammation scores in either univariate or multivariate analyses, findings consistent with prior studies (28). The only correlation found by investigators in Korea (29, 38) was between gastric juice ph and H. pylori density, confirming our observations. These findings suggest that the relationship between the degree of H. pylori colonization and subsequent host response is complex and predominantly host specific. In conclusion, we have extended earlier findings indicating that carriage of caga strains, as determined by PCR or serology, has a substantial impact on the extent of gastric inflammation and, consequently, on the risk of upper gastrointestinal disease. In contrast, gastric juice antibodies to H. pylori WC and CagA antigens do not appear to have an independent effect on either inflammation or disease, and their determination may better reflect the serum, rather than the secretory, immune responses to H. pylori. ACKNOWLEDGMENTS This work was supported in part by the Medical Research Service of the Department of Veterans Affairs and by RO1 DK53707, RO1 DK58587, and RO1 CA77955 from the National Institutes of Health. REFERENCES 1. Atherton, J. C., K. T. Tham, R. M. Peek, Jr., T. L. Cover, and M. J. Blaser Density of Helicobacter pylori infection in vivo as assessed by quantitative culture and histology. J. Infect. Dis. 174: Aucher, P., M. L. Petit, P. R. Mannant, L. Pezennec, P. 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