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challenged mice
April 1998 • G3830 POST-IMMUNIZATION GASTRITIS IN HELICOBACTER IMMUNIZED/ CHALLENGED MICE. T. G. Blanchard, $. J. Czi0n, R. Redline, N. Sigmund, and J. G. Nedrud. Case Westeru Reserve University, Cleveland, OH.
Several laboratories have shown that while challenge of immunized mice results in protection from infection, challenged mice exhibit enhanced gastritis when compared to infection of non-immunized mice using the H. felis/mouse model. It is now possible to induce protective immunity in the recently described H. pylori/mouse model. To determine whether postimmunization gastritis is also a feature of the H. pylori mouse model, C57BL/6 and BALB/c mice were either infected with H. pylori or orally immunized 4 times weekly with 75 mg urease plus 10 pg cholera toxin. Immunized mice were challenged one week after immunization with 107 viable 1t. pylori. Three weeks following challenge mice were necropsied and examined for evidence of gastric inflammation and/or infection. Results: All immunized mice were protected from challenge with either H. pylvri or H. felis as determined by urease assay, culture, and/or Steiner's stain. However, immunized/challenged C57BL/6 mice exhibited significantly greater inflammation as compared to non-immunized challenged control mice. ! Group
Challenge Protection Semm IgG SerumIgA Inflammation (107 cfu) lOglQ lOglQ (score 0-5) BALB/c naive H.pylori 0/6 2.0 ± .91 1.0 + .41 1.3 ± .82 BALB/c immune H.pylori 8/8 5.9 ± 1.3 2.4 ± .38 1.7 ± .52 C57BL/6 naive H.pylori 0/6 3.1 ± .75 0.5 ± 0.0 .67 ± .82 H.pylori immune i H.pylori 8/8 6.7 ± .88 2.7 ± .69 2.0 ± .89* H.pylori naive H. felis 0/6 3.4 ± .48 .75 ± .29 3.0 ± 1.1 H.pylori immune H. felis 8/8 7.7 ± .52 3.0 ± .63 4.5 ± .55* *P < 0.05 compared to correspondingnaive/challenged mice, student-sunpaired t test
Challenge of immunized C57BL/6 mice with H. pylori results in post immunization gastritis similar to that described for the H. felis mouse model. Based on previous H. felis protection studies this finding may also be due to residual (undetectable) low levels of H. pylori present within the gastric mucosa. • G3831 ATTENUATED SHIGELLA AS VECTORS FOR FOREIGN ANTIGEN DELIVERY: EFFECT OF virG MUTATIONS PREVENTING CELL TO CELL SPREAD ON SYTEMIC AND MUCOSAL IMMUNE RESPONSES. P. BIoQm, R. Russell, D. Blake, T. Bowen, F. Noriega, E. Boedeker, Baltimore, MD; M. Le, W. Brown, Denver, CO.
Attenuated Shigella developed as oral vaccines may be useful vectors to deliver foreign antigens to the mucosal immune system since they target gut associated lymphoid tissue (GALT). Shigella with mutations in virG, which determines cell to cell spread, can target inductive sites in the Peyer's patch and multiply intracellularly but cannot polymerize actin as required to infect adjacent epithelial cells. To determine the effect of a virG mutation on the ability of a A aroA Shigellaflexneri 2a vaccine strain, CVD1201, to induce immune responses to an E. coli pihis antigen, we: 1) introduced plasmid pM5 encoding the AF/R1 pilus of rabbit diarrheal E. coli (RDEC-1) into Shigella flexneri 2a vaccine strains CVD1201 (AaroA) and CVD1203 (AaroA, AvirG) 2) documented pilus expression; and 3) tested the immunogenicity of the constructs in rabbits. Design: The transformed Shigellae (CVD1201/AFR1 and CVD1203/AFR1) were orogastrically administered in priming and booster doses of 109 CFU to two groups of 6 rabbits on day 1 and 14. Control groups of 6 animals received the vectors CVD1201 or CVD1203. Animals were sacrificed on day 26. Immunogenicity was assessed by measuring antiAF/R1 serum IgG, biliary IgA by ELISA and by quantitating anti-AF/R1 specific antibody secreting cells (ASC) in the gut lamina propria by immunohistochemistry. Results: Both transformed vaccine strains expressed AF/RI pili by agglutination and Western blot. Rabbits had no ill effects post vaccination. 6/6 Animals inoculated with CVD1201/AFRI but only 4/6 animals innoculated with CVD1203/AFR1 developed increasing serum IgG titers to AF/R1. All animals receiving CVD1201/AFR1 or CVD1203/AFR1 developed biliary IgA to the pilus as well as ASCs specific for AF/R1 in the lamina propria. The numbers of mucosal ASCs were less in animals immunized with CVD1203/AFR1. Rabbits immunized with control strains CVD1201 and CVD1203 did not develop serologic, biliary or mucosal immunity to AF/R1. Conclusions: Shigellae are effective vectors for delivering foreign antigens to the mucosal and systemic immune system. Vaccines which remain in the Peyer's patch, such as virG mutants, may have diminished systemic immunogenicity, while maintaining mucsoal immunogenicity. • G3832 SUBSTANCE P (SP) REGULATES SOMATOSTATIN (SOM) EXPRESSION IN INFLAMMATION. Arthur Blum, David Elliott, Ahmed Metwali, Jie Li, Khurram Qadir and Joel Weinstock Division of Gastroenterology, Dept. of Int. Med., Univ. of Iowa, Iowa City, IA. Introduction: Poorly controlled granulomatous inflammation caused by excess production of Thl-type cytokines like IFN-7 leads to intestinal injury
Immunology, Mierobiology~ and Inflammatory Disorders A935
in CD. SP and SOM are short polypeptides present throughout the gastrointestinal tract. There is a SP/SOM immunoregulatory circuit that modulates Thl responses at mucosai surfaces. SP enhances, while SOM decreases IFN-T secretion. SP and SOM networks are altered in IBD. Macrophages, induced by various inflammatory mediators, make large amounts of SOM. The goal of this study was to determine if there also are regulatory factors that can limit macrophage SOM expression. Results: It was discovered that SP is a powerful regulator of SOM synthesis. Murine splenocytes cultured with LPS, IFN-'f or IL-10 for 4h strongly expressed SOM mRNA. Splenocytes exposed to SP at 10-9 M or higher prevented LPS, IFN-7 and IL-10 induction of SOM mRNA (Figure 1). The inhibition at 10-9 M was >95% as shown by quantitative PCR. Also, splenocyte SOM content decreased from 3850 to <10pg/sample following SP exposure, lmmunohistochemistry identified splenic macrophages as the source of the SOM. Mice infected with S. mansoni form granulomas in the liver and intestines resulting from deposition of parasite eggs in these organs. The granulomas contain macrophages that make SOM constitutively. SP at 10.8 M decreased SOM mRNA expression >90% in dispersed granuloma cells cultured 4h. Specific SP receptor antagonists blocked SP suppression of SOM expression showing that an authentic SP receptor mediated the regulation. Other studies showed that IL-4 is a natural antagonist of the SP effect.
It is concluded that: 1) Murine macrophages make large amounts of SOM in response to cytokines. 2) SP inhibits both SOM induction and ongoing expression through interaction with a SP receptor. 3) IL-4 neutralizes the SP effect. The above findings signify that SP regulation of SOM expression is biologically relevant and potentially important in IBD. DK38327, DK02428, DK25295, CCFA, VAMC. • G3833 IMMUNOMODULATION OF HELICOBACTER FELLS COLONIZATION OF THE MOUSE STOMACH. J. Bo, M. Jordana, F. Smaill, D.P. Snider, R. Borojevic, D. Steele-Norwood, R.H. Hunt, and K. Croitoru. Intestinal Diseases Research Program, Department of Medicine and Pathology, McMaster University, Hamilton Ontario. Background: Modulating helper T cell (Th) responses could influence host resistance or susceptibility to infection. Vaccines that prevent or eliminate Helicobacter infection in mice are dependent on mucosal adjuvants that might function by shifting the immune response toward Th2 predominant responses (IL-4, IL-6, 1L-10). On the other hand, it has been suggested that interferon-y (IFN) is important for the development of the gastric inflammation associated with Helicobacter infection. Aim: To test these hypotheses in the mouse model of Helicobacter infection, we used a replication-deficient human Adenovirus (RDA) to investigate whether RDA incorporated with raiL-10, which directs a Th2 response, could influence H. felis colonization in C57BL6 (B6) mice. We also examined the nature of H.felis infection in the IFN deficient, IFN-y-/- mice. Methods: Mice were fed 1-1.felis by gavage on three separate days, two days apart. Ten weeks after infection, B6 mice were injected in the hind limb with RDA alone or with RDA/mIL-10 twice at 5 day intervals. Mice were sacrificed and the tissue was prepared for histopathology. Gut washes and serum were collected for ELISA analysis of H. felis specific IgA, IgG1 and IgG2a responses. Results: All infected mice developed chronic inflammation by 8 weeks. Surprisingly, B6 mice treated with RDA alone showed a significant reduction in the number ofH. felis organisms in the gastric mucosa, while RDA-mIL-10 treated B6 mice remained well colonized. Significant differences in Ab responses were not detected in these two groups. IFN-y-/" mice showed a similar degree of gastric infection and inflammation as B6 mice. H. felis specific IgG2a was 5 times higher in B6 than in IFN-74- mice; while specific TgG1 was 2 times lower in B6 mice. Conclusions: We conclude that IFN-7 is not required for the development of H. )'blis-associated gastritis and that a predominance of IgG1 antibody in the H. felis response is not sufficient to eliminate H. fells infection. Furthermore, RDA infection alone leads to a significant reduction of established H. felis infection, but RDA infection combined with IL-10 release does not affect H. felis infection or inflammation. Whether the absence of IFN-7 or IL-10 in combination with RDA has shifted the Thl,ri'h2 balance remains to be determined. Funded by the Chedoke-McMaster Foundation.
Several laboratories have shown that while challenge of immunized mice results in protection from infection, challenged mice exhibit enhanced gastritis when compared to infection of non-immunized mice using the H. felis/mouse model. It is now possible to induce protective immunity in the recently described H. pylori/mouse model. To determine whether postimmunization gastritis is also a feature of the H. pylori mouse model, C57BL/6 and BALB/c mice were either infected with H. pylori or orally immunized 4 times weekly with 75 mg urease plus 10 pg cholera toxin. Immunized mice were challenged one week after immunization with 107 viable 1t. pylori. Three weeks following challenge mice were necropsied and examined for evidence of gastric inflammation and/or infection. Results: All immunized mice were protected from challenge with either H. pylvri or H. felis as determined by urease assay, culture, and/or Steiner's stain. However, immunized/challenged C57BL/6 mice exhibited significantly greater inflammation as compared to non-immunized challenged control mice. ! Group
Challenge Protection Semm IgG SerumIgA Inflammation (107 cfu) lOglQ lOglQ (score 0-5) BALB/c naive H.pylori 0/6 2.0 ± .91 1.0 + .41 1.3 ± .82 BALB/c immune H.pylori 8/8 5.9 ± 1.3 2.4 ± .38 1.7 ± .52 C57BL/6 naive H.pylori 0/6 3.1 ± .75 0.5 ± 0.0 .67 ± .82 H.pylori immune i H.pylori 8/8 6.7 ± .88 2.7 ± .69 2.0 ± .89* H.pylori naive H. felis 0/6 3.4 ± .48 .75 ± .29 3.0 ± 1.1 H.pylori immune H. felis 8/8 7.7 ± .52 3.0 ± .63 4.5 ± .55* *P < 0.05 compared to correspondingnaive/challenged mice, student-sunpaired t test
Challenge of immunized C57BL/6 mice with H. pylori results in post immunization gastritis similar to that described for the H. felis mouse model. Based on previous H. felis protection studies this finding may also be due to residual (undetectable) low levels of H. pylori present within the gastric mucosa. • G3831 ATTENUATED SHIGELLA AS VECTORS FOR FOREIGN ANTIGEN DELIVERY: EFFECT OF virG MUTATIONS PREVENTING CELL TO CELL SPREAD ON SYTEMIC AND MUCOSAL IMMUNE RESPONSES. P. BIoQm, R. Russell, D. Blake, T. Bowen, F. Noriega, E. Boedeker, Baltimore, MD; M. Le, W. Brown, Denver, CO.
Attenuated Shigella developed as oral vaccines may be useful vectors to deliver foreign antigens to the mucosal immune system since they target gut associated lymphoid tissue (GALT). Shigella with mutations in virG, which determines cell to cell spread, can target inductive sites in the Peyer's patch and multiply intracellularly but cannot polymerize actin as required to infect adjacent epithelial cells. To determine the effect of a virG mutation on the ability of a A aroA Shigellaflexneri 2a vaccine strain, CVD1201, to induce immune responses to an E. coli pihis antigen, we: 1) introduced plasmid pM5 encoding the AF/R1 pilus of rabbit diarrheal E. coli (RDEC-1) into Shigella flexneri 2a vaccine strains CVD1201 (AaroA) and CVD1203 (AaroA, AvirG) 2) documented pilus expression; and 3) tested the immunogenicity of the constructs in rabbits. Design: The transformed Shigellae (CVD1201/AFR1 and CVD1203/AFR1) were orogastrically administered in priming and booster doses of 109 CFU to two groups of 6 rabbits on day 1 and 14. Control groups of 6 animals received the vectors CVD1201 or CVD1203. Animals were sacrificed on day 26. Immunogenicity was assessed by measuring antiAF/R1 serum IgG, biliary IgA by ELISA and by quantitating anti-AF/R1 specific antibody secreting cells (ASC) in the gut lamina propria by immunohistochemistry. Results: Both transformed vaccine strains expressed AF/RI pili by agglutination and Western blot. Rabbits had no ill effects post vaccination. 6/6 Animals inoculated with CVD1201/AFRI but only 4/6 animals innoculated with CVD1203/AFR1 developed increasing serum IgG titers to AF/R1. All animals receiving CVD1201/AFR1 or CVD1203/AFR1 developed biliary IgA to the pilus as well as ASCs specific for AF/R1 in the lamina propria. The numbers of mucosal ASCs were less in animals immunized with CVD1203/AFR1. Rabbits immunized with control strains CVD1201 and CVD1203 did not develop serologic, biliary or mucosal immunity to AF/R1. Conclusions: Shigellae are effective vectors for delivering foreign antigens to the mucosal and systemic immune system. Vaccines which remain in the Peyer's patch, such as virG mutants, may have diminished systemic immunogenicity, while maintaining mucsoal immunogenicity. • G3832 SUBSTANCE P (SP) REGULATES SOMATOSTATIN (SOM) EXPRESSION IN INFLAMMATION. Arthur Blum, David Elliott, Ahmed Metwali, Jie Li, Khurram Qadir and Joel Weinstock Division of Gastroenterology, Dept. of Int. Med., Univ. of Iowa, Iowa City, IA. Introduction: Poorly controlled granulomatous inflammation caused by excess production of Thl-type cytokines like IFN-7 leads to intestinal injury
Immunology, Mierobiology~ and Inflammatory Disorders A935
in CD. SP and SOM are short polypeptides present throughout the gastrointestinal tract. There is a SP/SOM immunoregulatory circuit that modulates Thl responses at mucosai surfaces. SP enhances, while SOM decreases IFN-T secretion. SP and SOM networks are altered in IBD. Macrophages, induced by various inflammatory mediators, make large amounts of SOM. The goal of this study was to determine if there also are regulatory factors that can limit macrophage SOM expression. Results: It was discovered that SP is a powerful regulator of SOM synthesis. Murine splenocytes cultured with LPS, IFN-'f or IL-10 for 4h strongly expressed SOM mRNA. Splenocytes exposed to SP at 10-9 M or higher prevented LPS, IFN-7 and IL-10 induction of SOM mRNA (Figure 1). The inhibition at 10-9 M was >95% as shown by quantitative PCR. Also, splenocyte SOM content decreased from 3850 to <10pg/sample following SP exposure, lmmunohistochemistry identified splenic macrophages as the source of the SOM. Mice infected with S. mansoni form granulomas in the liver and intestines resulting from deposition of parasite eggs in these organs. The granulomas contain macrophages that make SOM constitutively. SP at 10.8 M decreased SOM mRNA expression >90% in dispersed granuloma cells cultured 4h. Specific SP receptor antagonists blocked SP suppression of SOM expression showing that an authentic SP receptor mediated the regulation. Other studies showed that IL-4 is a natural antagonist of the SP effect.
It is concluded that: 1) Murine macrophages make large amounts of SOM in response to cytokines. 2) SP inhibits both SOM induction and ongoing expression through interaction with a SP receptor. 3) IL-4 neutralizes the SP effect. The above findings signify that SP regulation of SOM expression is biologically relevant and potentially important in IBD. DK38327, DK02428, DK25295, CCFA, VAMC. • G3833 IMMUNOMODULATION OF HELICOBACTER FELLS COLONIZATION OF THE MOUSE STOMACH. J. Bo, M. Jordana, F. Smaill, D.P. Snider, R. Borojevic, D. Steele-Norwood, R.H. Hunt, and K. Croitoru. Intestinal Diseases Research Program, Department of Medicine and Pathology, McMaster University, Hamilton Ontario. Background: Modulating helper T cell (Th) responses could influence host resistance or susceptibility to infection. Vaccines that prevent or eliminate Helicobacter infection in mice are dependent on mucosal adjuvants that might function by shifting the immune response toward Th2 predominant responses (IL-4, IL-6, 1L-10). On the other hand, it has been suggested that interferon-y (IFN) is important for the development of the gastric inflammation associated with Helicobacter infection. Aim: To test these hypotheses in the mouse model of Helicobacter infection, we used a replication-deficient human Adenovirus (RDA) to investigate whether RDA incorporated with raiL-10, which directs a Th2 response, could influence H. felis colonization in C57BL6 (B6) mice. We also examined the nature of H.felis infection in the IFN deficient, IFN-y-/- mice. Methods: Mice were fed 1-1.felis by gavage on three separate days, two days apart. Ten weeks after infection, B6 mice were injected in the hind limb with RDA alone or with RDA/mIL-10 twice at 5 day intervals. Mice were sacrificed and the tissue was prepared for histopathology. Gut washes and serum were collected for ELISA analysis of H. felis specific IgA, IgG1 and IgG2a responses. Results: All infected mice developed chronic inflammation by 8 weeks. Surprisingly, B6 mice treated with RDA alone showed a significant reduction in the number ofH. felis organisms in the gastric mucosa, while RDA-mIL-10 treated B6 mice remained well colonized. Significant differences in Ab responses were not detected in these two groups. IFN-y-/" mice showed a similar degree of gastric infection and inflammation as B6 mice. H. felis specific IgG2a was 5 times higher in B6 than in IFN-74- mice; while specific TgG1 was 2 times lower in B6 mice. Conclusions: We conclude that IFN-7 is not required for the development of H. )'blis-associated gastritis and that a predominance of IgG1 antibody in the H. felis response is not sufficient to eliminate H. fells infection. Furthermore, RDA infection alone leads to a significant reduction of established H. felis infection, but RDA infection combined with IL-10 release does not affect H. felis infection or inflammation. Whether the absence of IFN-7 or IL-10 in combination with RDA has shifted the Thl,ri'h2 balance remains to be determined. Funded by the Chedoke-McMaster Foundation.