Real-time fluorogenic RT-PCR analysis of 12 cytokines in Helicobacter pylori colonized patients

Real-time fluorogenic RT-PCR analysis of 12 cytokines in Helicobacter pylori colonized patients

A330 AGA ABSTRACTS GASTROENTEROLOGY Vol. 118, No, 4 1792 1794 ASSESSMENT OF ANTIBODY STATUS OF MICE INFECTED WITH HELICOBACTER PYLORI PRE· AND POS...

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A330 AGA ABSTRACTS

GASTROENTEROLOGY Vol. 118, No, 4

1792

1794

ASSESSMENT OF ANTIBODY STATUS OF MICE INFECTED WITH HELICOBACTER PYLORI PRE· AND POST TREATMENT. Linda M. Best, Donna Hutchison, Sander 1. Veldhuyzen van Zanten,

REAL·TIME FLUOROGENIC RT·PCR ANALYSIS OF 12 CYTO· KINES IN HELICOBACTER PYLORI COLONIZED PATIENTS.

Queen Elizabeth II Health Sci Ctr, Halifax, NS, Canada; Dalhousie Univ, Halifax, NS, Canada. Representative animals are sacrificed to prove infection and eradication in trials using animal models to test the efficacy of antimicrobial agents. Aim: To discover whether the antibody response in mice can indicate infection and eradication without sacrifice. Methods: We assessed the antibody response in uninfected C57IBL6 mice and after infection with H. pylori prior to and following treatment with various regimens. One hundred ILl of blood was drawn from the saphenous vein into a vial, centrifuged, and frozen until tested by a previously validated flow cytometric immunofluorescence method. Polystyrene beads coated with H. pylori whole-cell antigen were added to 5 ILl of serum diluted I :20 in phosphate buffered saline, incubated at 31lC for 20 minutes, washed, added to goat anti-mouse IgG, incubated as above, washed, and analyzed using a flow cytometer. Comparative antibody levels were statistically generated. Results: In H. pylori-infected mice the average increase was 33 units. Four of 200 mice were below the cut-off for infection. The level decreased by an average of 24 units in successfully treated mice at four weeks, and increased by an average of 15 units in untreated and unsuccessfully treated mice. Conclusion: Antibody levels in the mouse readily indicate infection and response to treatment.

Mouse Anti·H. pylori IgGResponse Treatment Failure

Thomas Wigginton, Eric M. Osgard, Jon P. Kushner, Corinne Maydonovitch, William F. Blakely, Andre Dubois, USUHS; Dept of Medicine, Bethesda, MD; Applied Cellular Radiobiology Dept AFRRI, Bethesda, MD.

Helicobacter pylori induces intense inflammation in all colonized subjects and, in addition, causes peptic ulcer disease and/or gastric cancer in 10-15% of these subjects. It has been proposed that excessive inflammation is responsible for disease in those subjects. The goals of the present study were twofold: (1) to validate and use a novel assay to quantify the cytokine profile of colonized and non-colonized patients and (2) to determine whether cytokine mRNA expression is related to gastritis and/or H. pylori colonization. Six H. pylori positive patients with non-ulcer dyspepsia (4 positive by serology, 13C-UBT, CLO test, and culture; 2 by serology alone) were included. Esophagogastroduodenoscopy was performed before and after treatment, and biopsies were harvested and placed either on ice for culture, in 10% formalin for histology (Genta stain using Sydney grading criteria), or flash-frozen in liquid nitrogen within to seconds of sampling. From these frozen biopsies, total RNA was extracted, quantified by UV spectrophotometry, and reverse-transcribed to cDNA using random primers. Samples were analyzed by PeR with fluorogenic probes on human cytokine plates (Perkin Elmer Applied Biosystems) designed to quantify 12 human cytokine targets using the 5-fluorogenic nuclease Taqman TM assay. Cytokine targets, IL-l a, IL-I 13, IL-2, IL-4, IL-5, IL-8, IL-tO, IL-12p35, IL·12p40, IL-15, IFN'Y' and TNFa, were normalized to a control target sequence (18S rRNA). mRNA expression ofIL-lj3, IL-8, IL-tO, and TNFa was detected in 6 of6patients; IL-15 in 5 patients; IL-la and IFN'Y in 4 patients; and IL-2 in 3 patients. A significant direct correlation was observed between gastritis and the increased expression of IL-8, IL-tO, and TNFa (p<.05). A trend towards a decrease in expression of IL-8 and TNFa in the 2 patients positive by serology alone compared to the 4 patients positive by all tests also was observed. These data support the evidence of the direct relationship between histological gastritis and cytokines detected by molecular biology. These observations will need to be reevaluated when additional patients are enrolled, but they indicate that this novel, real-time quantitative PeR assay can automate the detection of multiple cytokines simultaneously. 1795 REFUTING THE DOGMA THAT INFLAMMATORY BOWEL DIS· EASES (IBD) ARE DISEASES OF HIGHER SOCIOECONOMIC STATUS: POPULATION·BASED STUDIES.

10'

10'

10

1793 COMPARISON BETWEEN ORAL AND NASAL IMMUNIZATION AGAINST H. PYLORI WITH CO·ADMINISTRATION OF RECOM· BINANT CHOLERATOXIN B SUBUNIT. Katsushi Watanabe, Takashi Joh, Katsuyuki Miyashita, Isami Todoroki, Nobuo Takahashi, Hideo Suzuki, Hiromi Kataoka, Makoto Sasaki, Kyouji Seno, Yoshifumi Yokoyama, Kunio Tochikubo, Makoto Itoh, Nagoya City Univ Med Sch, Nagoya, Aichi, Japan. Background and Aims: Choleratoxin (CT) is often used as an experimental mucosal immuno-adjuvant, while cr possesses the strong toxicity to human intestinal mucosa. We have previously reported that recombinant choleratoxin B subunit (rCTB), which was obtained from Bacillus brevis carrying CTB gene-inserted plasmid pNU212, can be used as a non-toxic adjuvant against H. pylori (Hp). However, it has not been clarified which route is best for the mucosal immunization against Hp with rCTB. In this study, we investigated the mucosal and systemic immuno-adjuvant activities of rCTB by oral and nasal immunization. Methods: Female C57IBL6 mice aged 7 weeks were used for this study. Mice were administered I mg of Hp sonicate plus 50 JJ.g of rCTB for oral immunization, or 40 JJ.g of Hp sonicate plus 10 ILg of rCTB for nasal immunization on days 0, 7, 14 and 21. Mice were starved and sacrificed on day 42. Blood and lavage fluid of stomach and small intestine were collected. Hp-specific IgG or IgA antibody titers in serum and lavage fluids were estimated using ELISA. Hp-specific IgE activity in serum was estimated by passive cutaneous anaphylaxis test. Results: When Hp was administered with rCTB by oral or nasal route, significant increases in IgG and IgA were detected (see the table). However, no Hp-specific IgE activity was detected, although such an IgE activity was detected in the mice in which Hp + CT were administered. Conclusion: I) In nasal as well as oral immunization, co-administration of CTB demonstrated significant adjuvant properties for stimulating IgG and IgA, but not IgE immune responses to Hp antigens. 2) Nasal immunization induced predominant IgG response in the stomach. 3) These results indicated that rCTB can be used as non-toxic and non-allergic mucosal adjuvant for vaccine against Hp. Immunogloblin responses after oral ornasal immunization with Hpplus rCTS serum IgG

IgA

stomach IgA IgG

2/6

0/6 4/6 0/4 4/4

0/6 4/6 0/4 4/4

positive I totalmice

oralimmunization oralimmunization nasal immunization nasal immunization

Hp only Hp +rCTS Hp only Hp +rCTS

6/6 0/4 4/4

0/6 4/6 0/4 0/4

small intestine IgA IgG

0/6 4/6 0/4

0/6 5/6

0/4

Charles N. Bernstein, Allen Kraut, James F. Blanchard, Patricia Rawsthorne, Nancy Yu, Randy Walld, Cameron Mustard, Univ of Manitoba, Winnipeg, MB, Canada. Background ffiD may impact on employment, education & marital status. It has long been held that ffiD patients are of a higher socioeconomic strata & more educated than the general population. We aimed to determine the influence of ffiD on work status, income, education level and marital status compared to population controls. Methods: 2 studies are reported herein. Study A: Questionnaire responses (n=2759) from the population-based Uof M ffiD Database (1996) were compared to employment, education, & marital status data from the 1996 National Population Health Survey (n=14,177 for Manitoba). Study B: we used the linked Manitoba Health Statistics Canada database (utilizes the 1986 Canada Census) & applied our administrative definition of ffiD. We extracted all working age (18-64 yrs) IBD patients (n=80) to be compared with the non-ffiD cohort (n=25,554). Labor force participants were either employed or actively seeking work. Results: Study A: Combining rates of unemployment & disability ffiD males and females, respectively, were more likely to be out of work in 1996 (12.5%,11.8%) than at diagnosis (5.8%, 6.5%) or than controls (8.4%, 7.2%) (p
1796 CHARACTERIZATION OF GENOMIC AND FUNCTIONAL VARIATION THROUGHOUT THE TNF GENE IN PATIENTS WITH ran Denise K. Bonen, Richard Ramos, Sanggyu Lee, Sarah Corradino, Heidi M. Britton, Barbara S. Kirschner, Steven R. Brant, Stephen B. Hanauer, Judy H. Cho, Univ of Chicago, Chicago, IL; Johns Hopkins Univ Sch of Medicine, Baltimore, MD. Background: Deletion of the AU-rich region in the 3' UTR of the tumor necrosis factor (TNF) gene increases expression in mice and results in an