Efficacy a Reduction of H Pylori Infection - Research Paper Example

Topic: Efficacy of vaccines-induced macrophages in reduction of H. pylori infection Name: Student ID: Institution: Tutor’s name: Due Date: Abstract This paper presents an experiment in which vaccination model was developed and to establish vaccination effectiveness of vaccines-induced macrophages in reduction of H. pylori infection in mice. It also involves a study of infiltrates by use of simple method-Immunohistochemistry. In another experiment, the experiment is done with the purpose of testing the original hypothesis that microphages are important for vaccine induced protection against H. pylori infection. Microphages are depleted from mice and the effect is examined. The study involves the use of murine model of chronic H. pylori infection to classify the role of macrophages in enhancing immune responses and gastritis and prevention of the bacterial burden in H. pylori vaccination. The expected observations are based on what was observed in the previous experiments and the purpose of experimental protocol is described. The results of the experiment show that there is a reduction in gastric pathology induced by H. pylori but there is no impact on bacterial load in the stomach. According to the data macrophages play an important role in reducing gastric inflammation during H. pylori infection. TABLE OF CONTENTS Abstract 2 1. Introduction 4 2. Materials and Methods 4 3. Results 7 3.1. Experiment MD1 7 3.2. Experiment MD2 7 3.3. Experiment MD4 10 4. Discussion 11 7. Tables, Figures and Legends 15 5. Acknowledgements 19 6. References 19 1. Introduction Helicobater pylori or H. pylori refers to a helix-shaped bacterium found in the stomach and that has a sort history of only three decades according to its identification by Robin warren and Barry Marshall, who were both Australian scientists. During the research on gastric ulcers and chronic gastritis, they came across the bacterium (Warren, 2000). During that time, both these conditions were associated with microbes and people in developing countries are more likely to be infected more than those in developed countries despite the little percentage of population in the world being infected by the bacterium in their gastrointestinal tracts. This bacterium has been explained to be a contributing factor towards development of the stomach’s natural ecology. The negative effect of H. pylori is that it has a large number of strains genomes but only three have been sequenced successfully (Blaser, 2006). For instance, a particular strain of genomes contains base pairs that can reach a tune of 1.7 million while the number of genes is estimated to be 1550. There is a current investigation on genetic composition of H.pylori and its working principle and its potential to contribute to the spread of gastric damage. 2. Materials and Methods During the experiment, six to eight week old mice were used during the first experiment. The experiment was divided into three groups: A vaccinated+ Challenged, B challenged, and C control. During the second experiment, it was divided into three groups Group A 4 boxes mice that were vaccinated on, after 35 days all mice in group A and the boxes in all group 4 naïve boxes were infected with SS1, while the remaining boxes were un infected. The 4th experiment involved standard vaccination protocol with liposome IV injections was done before and after the challenge, non-depleted control and liposome dosing. The first experiment involved a study of gastric infiltrates in details by looking at the number of neutrophils and macrophages that were present in the infiltrates. In the first week of the experiment, the weights of the tubes in which tissues were placed were measured and compared with the weights when the tubes were full of tissues. A total of 5 mice were used in the experiment and the weights of the tissue were obtained by subtracting the weight of the empty tube from that of the full tube. The number of infiltrates and macrophages were obtained and the standard deviations and mean weights of the tissues were obtained. Standard deviations were calculated and the existence of colonies in the dilution was measured. This experiment was repeated in the second and the third weeks by use of different set of specimens. In the second experiment, cell infiltrates were analyzed using the procedure in the first experiment but this was done in more details. Flow cytometry was used in this experiment for the purpose of examining and counting microscopic particles such as cells that are suspended in a stream of fluid and passing them through an electronic device that counts the suspended particles. This was important because it enabled simultaneous multi-parametric analysis of a large number of particles within a short time. The results of the experiment were used to predict the effect of H. pylori infection, or vaccination on cell population in the stomach. In order to achieve the objectives of this experiment, the cells were dissolved as suspensions in reagents that were passed through the cytometry. The experiment was used to determine the effect of vaccination on cell populations while the effects of the same were also examined on organs tested. In the third experiment, the purpose of the experiment was to determine whether macrophages are important in vaccine induced protection. This was achieved by depleting the mice of the macrophages before being used during the experiment and the effect on the parameters compared with the initial experiments. The working principle of liposomes to deplete macrophages was investigated and the impact of macrophages on colonization with H. pylori was investigated. In addition, the experiment investigated the effect of depleting macrophages on the cell population in the stomach. The experiment was carried out by selecting mice in treatment groups. The first treatment group involved Vaccinated and Challenged labeled A, the second treatment group was challenged only labeled B, the third treatment group was the control group labeled C, the fifth group was Macrophage depletion labeled C, vaccinated and challenged labeled dA while the last group was macrophage depletion and challenged labeled bB. In each of these groups, tissue weight was determined, the total number of cells in the bodies of the mice, the number of cells in the selected organs, the mean and standard deviation and samples from these mice were used in the cytometry analysis. Cl2MBP-liposome mediated macrophage depletion diminishes by twenty four days following infection. In the vivo macrophage depletion; macrophages were depleted by use of well-characterized liposome-mediated macrophage technique. It also involved injection of mice with (CIMDP)-loaded liposomes. During this experiment, H.pylori culture and infection of mice was done. The stomachs of mice were dissected to determine the number of CFU per gram of stomach tissue as previously described. Standard vaccination protocols, SLI3261pyz97 were then used for immunization during the experiments the experiment prior to infection with H.pylori. The efficacy of depletion of the proportion of F4/80+ macrophages, cytometry tests were done for the organs of each treatment groups. Figure 1. Shows squares which shows liposomes treated mice, while circles show non-treated mice. Horizontal bars in the figure show medians. Mann-Whitney analysis was used to do the test for p-values. 3. Results 3.1. Experiment MD1 The purpose of the experiment was to determine the vaccination protocol that was effective by examining gastric infiltrates in details compared with the previous studies. These enabled prediction of the result of the experiment based on what had been previously studied. In the first week of the experiment, tissue weight was found to be averagely 0.1593 grams while the mean weight of the infiltrates was 5.40 grams. The average log 10 per gram of tissue was obtained to be 6.03 and the average number of neutrophils and macrophages in the tissues was found to be 6.53. The standard deviation in the weights of neutrophils and macrophages in the tissues were found to be 0.73. 3.2. Experiment MD2 The results of experiment MD1 showed that there were sufficient number of neutrophils and macrophages in the sample that could be used in the cyometry tests. This allowed experiment MD 2 to be conducted with observable changes that enabled attainment of objectives of the study. The results of Flow cytometry showed enabled identification of the number of lymphocytes, number of cells, neutrophils and macrophages in the samples from mice that were used during the experiment. These results were based on the organs where they were obtained such as the stomach, mesenteric lining, spleen and paragastric lining. The cells for the flow cytometry were examined on a weekly basis. In the first week, it was found that the total amount of lymph per gram of sample in the stomach was 18.88 while the total amount of neutrophils as a percentage of a gram of a sample of reagent was 25%. The number of neutrophils was 40. The results of examination of the paragastric lining showed that the total number of neutrophils was 1 and the percentage of neutrophils per gram of the substance was 3%. The number of lymphocytes in the stomach was found to be 20447 while in the sample from the paragastric lining was 1129. The number of T-cells in the sample from the stomach was found to be 41% of the CD3 cells while that from paragstric lining was 38% of the sample. In the sample from the spleen, it was found that an avarege of 11867 events were reported in which the percentage of lymph per gram of the sample was 30.67 per cent while the percentage of neutrophils per gram of the sample was 30%. The number of neutrophils was 29 while an average number of lymphocytes per sample were 9042. The number of T cells per sample of CD3 cells was found to be 44% while the percentage of CD8 T cells as a percentage of CD3 cells was found to be 36%. The same experiment was repeated in the second and third weeks and it the number of cells before and after staining was determined by cytometry method. In the spleen, it was found that the number of cells in the spleen of the mice to the power of six was 55.8, in the stomach was 0.48, in the paragastric lining it was 0.06, in the mesenteric lining it was 3 while the standard deviations in the stomach, paragastric lining, mesenteric lining and the spleen were 0.04, 0.51, 50.12 and 28.78 respectively. The populations that were identified by use of the antibodies include single lymphocytes, CD3 cells in the T cells, the number of CD4 cells in the T cells and CD8 cells in the T cells. These cells were studied in all the organs that were involved in the study such as the stomach, mesenteric lining and the spleen. It was observed that vaccination resulted into no effect on cell population. This was done by comparing the number of cells at the beginning and end of the experiments. For instance, it was observed that the number of CD3 cells, CD4 cells and T cells remained constant during and after the cytometry tests.           MØ as % of Lymph../Gran.. Neutrophils as % of Lymph../Gran..   % CD3+ T cells CD4+ T cells (% of CD3+) CD8+ T cells (% of CD3+) DAY 7 STOMACH A   18.88 0.25 81.09 43.30 36.76 B   9.04 0.08 - - - C   10.44 0.11 - - -   DAY 7 PARAGASTRIC LN A   1.49 0.03 68.86 41.71 50.07 B   17.12 0.21 27.83 38.80 39.75 C   7.36 0.20 - - -   DAY 7 MESENTERIC LN A   0.94 0.07 72.51 46.61 47.46 B   0.73 0.01 67.22 50.78 44.51 C   0.73 0.00 72.65 51.14 44.91   Table 1. The number of neutrophils, CD3+ cells, CD4+T cells and CD8+ cells in various organs in mice The measurements of various levels of macrophages and neutrophils as a percentage of lymph per gram of the sample showed that there were a low percentage of neutrophils in the stomach with a score of 25%, in the paragastric lining; it was 3% while in the mesenteric lining it was 7%. Day 6 (06.07.09) Count (#) CFU Mouse number Sex Urease Tube full Tube empty Tissue weight 10-3. 10-4. 10-5.     A-1 F + 6.892 6.789 0.103 84 14 6 8.40E+04 1.40E+05 A-2 F ++ 6.971 6.896 0.075 591 128 22 5.91E+05 1.28E+06 A-3 F + 6.928 6.872 0.056 113 19 2 1.13E+05 1.90E+05 A-4 F - 6.915 6.831 0.084 18 0 0 1.80E+04 0.00E+00   B-1 F ++ 6.906 6.854 0.052 257 15 10 2.57E+05 1.50E+05 B-2 F ++ 6.920 6.841 0.079 0 0 0     B-3 F ++ 6.861 6.801 0.060 413 102 36 4.13E+05 1.02E+06 B-4 F ++ 6.947 6.909 0.038 29 0 0 2.90E+04   C-1 F ++ 6.806 6.753 0.053 73 16 0 7.30E+04 1.60E+05 C-2 F ++ 6.970 6.943 0.027 147 13 7 1.47E+05 1.30E+05 C-3 F ++ 6.896 6.835 0.061 64 19 3 6.40E+04 1.90E+05 C-4 F + 7.112 7.035 0.077 20 7 4 2.00E+04 7.00E+04 dA-1 F + 6.983 6.924 0.059 79 60 5 7.90E+04 6.00E+05 dA-2 F ++ 6.905 6.820 0.085 47 30 3 4.70E+04 3.00E+05 dA-3 F ++ 6.975 6.888 0.087 130 32 2 1.30E+05 3.20E+05 dA-4 F - 6.900 6.790 0.110 54 13 0 5.40E+04 1.30E+05 Table 2. Procedures analyzed during the assessment process 3.3. Experiment MD4 After 21 days of infection, five mice from the vaccinated-infected were killed. Longitidinal sections of gastric tissue were immunostained for neutrophils (red) with a conjugated anti-Ly6G monoclonal antibody; Helicobacter (green) with a FITC conjugated polyclonal anti-sera, and DAPI. Figure 2. represents these findings where the broken lines indicate basal and luminal surfaces of the gastric epithelium. Filled arrows pointing in the right direction indicate neutrophils while dotted arrows from right to left indicate areas of H.pylori colonization. It was observed that infected control animals showed high levels of H. pylori colonization at day 21 (A), strong neutrophil staining was observed in spleen that were used as a positive control (B), and minimal background was observed in tissues stained with secondary antibodies alone (C). 4. Discussion This study provided an extension of the previous observations regarding importance of cells in vaccine-induced immunity against H. pylori in mice model. Despite the observation that H. pylori antigen results into induction of the cells of the specimen to produce neutrophil activation factors and there are reduced number of neutrophils in gastric mucosa of mice, the processes through which cells contribute in the clearance of H. pylori remains unknown. It was also observed that immunized mice without mast cells had low bacterial load in gastric mucosa in comparison with 2 log CFU to that of a mouse that has not been immunized mouse at the 4th week following the challenge. The results with the H. pylori are not satisfactory but provide a demonstration of a blunting vaccine efficacy when there are no mast cells. These variations can be attributed to a number of differences between different strains of H.pylori in addition to differences in inflammatory responses resulting from the two species (DeLyria, Redline, and Blanchard, 2009). During the process of determining tissue weight, the weights of empty test tube was subtracted from that of a full test tube to It was found that the tissue weight ranged from 0.103 to 0.27. This shows that the experiment was procedural before the final result was obtained. The CFU values ranged from 0.00E+ 0.00 to 6.00E+0.4. mean weights of the tissues were found and log mean per base 10 of the weight was determined. Mean weight and standard deviation in the weights of the tissues were determined and the highest value was found to be 4.70 while the lowest measured 4.5 grams. Groups A of the experiment involved experiments using mice that had been vaccinated and challenged. It was found that there was reduced number of H. pylori bacteria in them during vaccination. The second group did not involve any initial vaccination or vaccination despite being exposed to H.pylori bacteria. There was a high growth of the bacteria in the organs such as stomach and the spleen. This can be explained by the fact that when vaccination is done against the bacteria, it results into a reduction of the severity of their existence. The third group of the experiment involved experimenting with a control group. In this group, there was no subjection of the tissues with macrophages, however, the fourth group and fist the groups were vaccinated against the bacteria. It was found that there were larger number of macrophages in the groups that were not immunized against the ‘increased in number in the tissues compared with those who were immunized. Mast cells result from CD34+ multipotent hematopoietic bone marrow cells that are located in almost all tissues in the body, where final maturation process takes place in response to local factors. Activation of mast cells takes place through cross-linking of IgE antibodies resulting in degranulation and production of histamine in addition to other bioactive mediators that have impacts on the environment such as heparin in addition to other cytokines and chemokines. There has been recent expansion of the role of mast cells in immune response by including a direct response to microbial antigens through other receptors and CD8 cells. Such circumstances have resulted into production of appropriate mediators that enhance and complement effector-cell immune systems (Kleanthous and Monath, 1998). In response to introduction of H. pylori, mast cells have assumed higher densities in gastric mucosa in biopsy specimens that are infected with H.pylori. Since it is usually difficult to eradicate H. pylori without immunization of the mice, an increase in the number of mast cells and activity has the potential to result from T-cell signaling as a result of challenge of immunized mice. The data from the experiments in this paper show that mice that are deficient of mast cells have a greater than a 2-log reduction in H. pylori CFU in comparison with the CFU for mice that have not been immunized. Thus, it can be said that the function of mast cells in vaccine-induced H.pylori immunity is to activate neutrophils. When H.pylori antigens are used, they induce the production of TNF-α chemokines that are possible inducers of neutrophil activation. TNF- α that result from mast cells contributes significantly towards increased influx of neutrophils into areas that are affected by bacteria. Infected sites containing mast cells have been reported to have an impact on recruitment of neutrophils (Galli and Wershil, 1996). This experiment demonstrated that mice that had reduced number of mast cells had reduced levels of inflammation in addition to low number of TNF- α in comparison to the wild type mice in the model for immunization. According to the previous data, recruitment of neutrophils to the gastric mucosa is necessary for protection of the H.pylori. TNF- α is described as a factor for immune protection produced by mast cells for the function of recruiting neutrophils to bacterium-infected tissues. This study provided an investigation of CD4+ and CD4+ T function in vaccinated mice and compared them with gastric infiltrates for those that were in circulation in the stomach, spleen, mesenteric lining and lymph nodes. It was found that there was an increase in the number of CD4nin the stomach of vaccinated mice in addition to decreased ratio of T subpopulation of CD4+ cells in the stomach (Ismail, Fick, Zhang, Lynch and Berg, 2003). According to these data, vaccination resulted into low rates of suppression of CD4+ cell population and function, and results into a prediction that these cells are more likely to have a higher proliferative capacity. In this experiment, H.pylori vaccinated mice model the reduction of mRNA in the organs of immunized mast-cell mice provides an explanation for the reduction in neutrophils in the lamina proporia. Cytokines are secreted by the mast cells in response to T-cells and B-cells growth and function such as IL-3, -4, -5, -13, -16 and TNF- α.This results into a presentation of the evidence that shows that when mast cells are absent in mice, there is no impact on inductive phase of the anti-H. pylori immune response following immunization. It is postulated that T cells and mast cells undergo interactions either directly or indirectly in the organs following bacterial introduction for the purpose of promoting effector mechanisms that enable eradication of H.pylori infection. When mast cells are activated, T-cell migration is induced either directly or by the induction of adhesion molecule expression in the endothelial cells.The activated T-cells has the capability to influence recruitment of mast cells in the organs of the mice. Generally, these experiments show that mast cells while not significant for vaccination immune protection against H.pylori, have a contributing factor to protection of immunized mice. It is also demonstrated that the T-cells recall response to vaccination is not affected by absence of mast cells. The production capability of production of proinflammatory cytokines in response to H.pylori antigens is an indication that mast cells have the capability to participate in promotion of protective inflammation. 7. Tables, Figures and Legends Figure 1. Efficacy of depletion and the proportion of macrophages in the spleen of each treatment group Figure 2. Association between infiltrating neutrophils and H. pylori colonization in the Figure 2. Antibodies-Flow cytometry Supplier Antibody Clone Catalogue No Dilution BD Pharmingen PE-Cy7: Hamster α mouse CD3 145-2C11 552774 1:100 BD Pharmingen APC: Hamster α mouse CD3 145-2C11 553066 1:300 BD Pharmingen PE: Rat α mouse CD4 GK1.5 553730 1:500 BD Pharmingen PerCP: Rat α mouse CD8a 53-6.7 553036 1:500 BD Pharmingen PE: Rat α mouse CD8a 53-6.7 553033 1:500 BD Pharmingen APC-Cy7 Rat α mouse CD11b M1/70 557657 1:300 eBiosciences APC:Rat α mouse F4/80 BM8 17-4801-82 1:300 BD Pharmingen Rat $ mouse CD16/CD32-Mouse BD FcBlock 2.4 G2 55142 1:1000 BD Pharmingen PE: Rat α mouse Ly6G 1A8 551461 1:500, 1:50 R $ D Systems Polyclonal Goat $ mouse LeptinR - aF 97 1:100, 1:50 Jackson Immuno Research Laboratories Inc. Cy2: Donkey α goat IgG (H+L) - 705-225-147 1: 400, 1:200 Figure 3. Standard vaccination protocol with liposome injections IV immediately before and after challenge Day 0 14 28 30 35 5 days post Hp 42 14 days post Hp 56 Day 21 post ? Day 42 post? A Vaccinate SL3261pyz97 H. pylori challenge 4 8 8 B H. pylori challenge 4 8 8 C Vaccinate SL3261 Carrier Mock challenge 4 8 8 A deplete Vaccinate Liposomes iv Liposomes iv H. pylori challenge Kill 4 + Liposomes Iv for rest Liposomes iv 8 8 B deplete Liposomes iv Liposomes iv H. pylori challenge Kill 4 + Liposomes Iv for rest Liposomes iv 8 8 C deplete Vaccinate SL3261 Carrier Liposomes iv Liposomes iv Mock challenge Kill 4 + Liposomes Iv for rest Liposomes iv 8 8 Figure 4. CFU data used during the experiment Count CFU Tube full Tube empty Tissue weight 10^3 10^4 10^5     7.0826 6.9444 0.1382 4 2 0 4.00E+03 2.00E+04           0     6.9364 6.8134 0.1230 0 0 0 0.00E+00 0.00E+00         0 0   0.00E+00 6.9436 6.8112 0.1324 1 0 0 1.00E+03 0.00E+00           0     6.907 6.8316 0.0754 9 1 0 9.00E+03 1.00E+04         1 0   1.00E+04 7.0494 6.8896 0.1598 2 0 0 2.00E+03 0.00E+00           0     5. Acknowledgements This research acknowledges the contribution of the National Hospital for provision of facilities and reference materials for conducting the study. 6. References Akhiani, A. A., J. Pappo, Z. Kabok, K. Schon, W. Gao, L. E. Franzen, and N. Lycke. 2002. Protection against Helicobacter pylori infection following immunization is IL-12-dependent and mediated by Th1 cells. J. Immunol. 169:6977-6984.Aug;8(8):1037--‐49. Becher,D.,Deutscher,M.E.,Simpfendorfer,K.R.,Wijburg,O.L.,Pederson,J.S.,Lew,A.M.,etal. 2010.Local recall responses Blanchard, T. G., J. C. 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