抗幽门螺杆菌 z-bio 2021-07-14 696

　　Determine if systemic immunity to Helicobacter pylori is available for approved personal use. The efficacy of a vaccine combining Helicobacter pylori antigen and aluminum hydroxide (AlOH3) was evaluated in a mouse model of Helicobacter pylori infection. Antigen and -AlOH3 immunization can induce interleukin 5 secretion, antigen-specific T cells and immune antigens. ELISPOT determined that complete Freund's adjuvant induces type II interferon secretion and antigen-specific T cells. The protective effects of the two immune responses were evaluated by culturing two histological gastric biopsy specimens, because the bacteria were not confirmed after encountering Helicobacter pylori. The defense mechanism does not rely on antibodies. The use of antibody-deficient MMT mice (immunoglobulin knockout mice) and CD4 + spleen T cells from immunized mice are sufficient to deliver protective immunity to immunocompromised receptors. ...This result indicates that there are alternatives or potentially more strategies for the rapid development of Helicobacter pylori vaccines.

　　Helicobacter pylori, the most common pathogen in the world, is an extracellular bacteria that can infect the gastric mucosa, causing gastritis and peptic ulcers. It is generally believed that infectious diseases are mainly seen in young children. Therefore, the use of preventive vaccines can prevent infants from being infected with Helicobacter pylori and prevent long-term vaccination of adults. The vaccination strategy is mainly oral and nasal to induce mucosal immunity. These immunizations require the use of unsafe bacterial exotoxin adjuvants for humans. Recently, it has been reported that systemic immunity is a possible means of inducing and protecting mouse Helicobacter pylori immunity. The authors put forward a view that the contribution of Th1-type immune response and Helicobacter pylori-related response to gastric cancer pathology is necessary for inducing immunity. The recommendation is based on the analysis of immunoglobulin (IgG) classes and does not directly measure cytokines. Some reports have confirmed that this protection can be mediated by type 2 immunity. Use parenteral immune adjuvants to study this more direct method and use the ELISPOT method for detection. This produces a highly polarized Th1 or Th2 response. This study investigated whether the systemic immunity of aluminum hydroxide adjuvant (AlOH3) can induce protective Th2CD4T cell-mediated immunity in the absence of antibodies. AlOH3 induced immunity has great advantages in the development of Helicobacter pylori vaccine. This is because AlOH3 is approved for human use. Its safety, stability and low cost can accelerate the large-scale development and application of human Helicobacter pylori.

　　Materials and methods: The mice were purchased from Jackson Laboratory and raised without specific pathogens. The mice used were C57BL/6 or C57BL/6J-Rag1tm1Mom (rag1/) lacking mature T and B lymphocytes, or C57BL/6-Igh-6tm1Cgn (mMT) lacking mature B cells. In other words, it cannot produce antibodies.

　　Bacteria H. felis was isolated from cat stomach biopsy specimens in our laboratory. The Helicobacter pylori strain HPM6 was isolated from human gastric biopsy specimens and adjusted in our laboratory through long-term passage of mice. The PCR detection of cagA confirmed that HpM6 and C57BL/6 mice inoculated with HpM6 can cause chronic infection, which lasts for 12 months in 100-infected mice. Two types of Helicobacter were identified by colony morphology, cell morphology, Gram stain, urease, catalase, oxidase, etc.

　　Bacteria grow on solid medium (blood agar) and are suspended in Brinell broth medium.

　　Mouse immunity and pathway: Helicobacter pylori lysate, we and others have proven through oral route that this is an effective experimental vaccine antigen. Ovalbumin (OVA) was purchased from Sigma Chemical Co., Ltd., and AlOH3 was purchased from Pierce. Complete Freund's adjuvant (CFA) is an incomplete Freund's adjuvant made by mixing Mycobacterium tuberculosis H37RA (Difco Laboratories) with 1 mg/mL. The antigen and adjuvant were mixed in a 1:1 aqueous solution, and each mouse was injected intraperitoneally with 100 mg of antigen and 100 ml of emulsion on day 0. On the 28th day, 0.5 mL broth containing 1 × 107 cfu bacteria was orally administered by gavage through a gastric cannula through an 18-gauge needle. Determine the number of bacteria by using the previously established growth curve to set the optical density to 450 nanometers. As an isolate, H. felis is difficult to grow and reproduce. The use of standard absorbance values is determined according to the growth curve of Helicobacter pylori, as described in other documents.

　　Vaccine titer measurement: 28 days later, mice were infected with Helicobacter pylori through silver staining of the tissue. The animals were euthanized by the carbon dioxide method. The elongated tissue is surgically moved from the duodenum to the cardia. The tissue was fixed with 10 aldehyde buffer and histological examination was performed. Several parts of each mouse sample were stained with silver stain to help distinguish Helicobacter pylori and H. ferris from the position and morphology of the bacteria. Silver staining did not detect Helicobacter pylori in the tissues, confirming that the mice were immune protected. In addition, gastric biopsy specimens were cultured from mice infected with Helicobacter pylori and confirmed the presence of bacteria. A sample containing Helicobacter pylori was surgically removed from the gastric antrum and mixed with 200 ml of broth medium. Spread the homogenate on 100 mL of medium containing 7 blood. After 96 microaerobic cultures, the immune defense is determined by samples of mice immunized with uncultured bacteria. In the presence of bacteria, the colony morphology of Helicobacter pylori, Gram staining and the production of urease, catalase and oxidase determine the bacteria. The cultivation of H.felis is unreliable and cannot be operated. H.felis grows in pieces, not independently.

　　Pathological evaluation: Similar to other documents, the intensity of inflammation in the longitudinal section of mouse stomach curvature is evaluated. Some include the full length of the antrum and mucous glands in the fundus of the stomach. The inflammation grade of the pyloric sinus is 0-3, and the inflammation grade of the fundus is 1-10. The total score for each mouse is a linear range of depth (focal, multifocal, patchy or diffuse) (in the superficial and/or basal layer, submucosa or submucosa or muscle layer) and features of inflammatory infiltration. It is defined by changes in the tissue structure of the infiltrating cell type.

　　Using: mouse T cell CD4 subgroup column kit to purify CD4 T cells, using tail vein method to inject 10 million cells into mice. This number has been established and used in research in the field of autoimmunity.

　　enzyme immunoassay (ELISPOT) prepares a single cell suspension from the spleen. Inoculate 1 x 106 cells in serum-free HL-1 medium containing 1 mM glutamine per well with or without Helicobacter pylori antigen. The final concentration is 5 mg/ml. These cultures were added to the enzyme-binding immunospot plate, and PBS specifically captured interferon or interleukin 5, R46-A2 (4 mg/ml) or TRFK5 (5 mg/ml), respectively. At room temperature, the cells were blocked with PBS containing 1 serum albumin for 1 hour, and the cells were eluted with PBS 4 times before starting the 24 hour cell culture. The cells were then removed by the elution method, 1 mg/mL XMG1.2-HRP as IFN-g and 4 mg/mL TRFK4 as IL-5 were added to monitor antibodies and the plate was incubated. For IL-5, add anti-IgG2a-HRP and continue incubating for 2 hours. By adding 3-amino-9-ethyl-carbazole, you can see the antibody in the plate. Use Series 1 Immunospot Image Analyzer to evaluate the results.

　　Statistical analysis: The analysis of ELISPOT between experimental groups is determined by analysis of variance. Fisher's exact test was used to evaluate the infection or non-infection of the immunized mice.

　　Result: The mouse model of Helicobacter pylori infection and immunity uses human pathogens or H. felis isolated from cats. Unlike Helicobacter pylori, using C57BL/6 mice, H. felis caused inflammatory lesions of the mouse gastric mucosa, similar to human gastritis. In addition, according to the tissue sections of the mouse stomach, H. felis has a more serious infection. Unlike Helicobacter pylori, it is mainly located at the junction of the fundus. It mainly grows and regenerates in the antrum and fundus of the stomach. C57BL/6 mice were immunized with cat H. felis or Helicobacter pylori antigen and aluminum hydroxide AlOH3 or complete Freund's adjuvant CFA emulsifier. Our recent research suggests that these adjuvants may trigger type 2 and type 1 immune responses. After 14 days, the type 1 and type 2 cytokines (IFN-γ, IL-5, respectively) were measured by using the enzyme-binding immunospot test (ELISPOT), and the spleen cells of these mice had Helicobacter pylori antigen specificity. Reply. Mouse cells immunized with antigen and emulsified with aluminum hydroxide may produce IL-5 lacking IFN-r, and the number of IL-5 producing cells is higher than the number of antigen-specific IFN-r, both of which are immunized by Helicobacter pylori. antigen. After immunization with the antigen and complete Freund's adjuvant group, the opposite number of cytokines, that is, high IFN-r, was observed. In addition, the serum of mice immunized with complete Freund's adjuvant CFA also had a higher titre of IgG2a antibody. In the case of antigen emulsifiers, the type 2 immune response induced by aluminum hydroxide is the type 1 immune response stimulated by complete Freund's adjuvant, based on the relative number of cells produced and the distribution of the immune response of these antibody subtypes. Next, we tested whether induction of type 2 or type 1 Helicobacter pylori antigen immunity can protect the intestinal mucosa from Helicobacter pylori infection. C57BL/6 mice were immunized with Helicobacter pylori or feline hepatitis emulsified with aluminum hydroxide or complete Freund's adjuvant. The injectable adjuvant of the control group mice contained irrelevant control protein, ovalbumin, or remained unimmunized. 28 days and 28 days later, 1×107 Helicobacter pylori was orally administered orally, and the number of bacteria in the gastric mucosa was visually checked by silver staining. The presence or absence of Helicobacter pylori is determined by the bacterial culture method. In this study of fully immunized mice, it does not matter which adjuvant provides very good protection. Observe that there is no Helicobacter pylori in the silver-stained tissue.

　　However, 12 out of 14 control mice were infected with Helicobacter pylori. Helicobacter pylori was injected into 7 out of 8 mice. No infection after injection of aluminum hydroxide. The protective effect of the adjuvant aluminum hydroxide and complete Freund's adjuvant is very important. Mouse H. felis is more severely infected than Helicobacter pylori. Twenty-eight out of 29 mice were infected and had a strong attachment of bacteria to their stomachs. There are an average of 86 infected glands in each stomach. No bacteria were detected in mice using H.feli antigen aluminum hydroxide adjuvant. The use of complete Freund's adjuvant and Helicobacter pylori emulsifier also had a protective effect (5 out of 21 people were infected). We and other studies have found that effective mucosal vaccination strategies require the use of cholera toxin and adjuvants. The use of aluminum hydroxide and complete Freund's adjuvant and antigen are also associated with effective mucosal vaccination strategies. In contrast to the few post-emulsification infections of H. felis in sham-immunized mice with complete Freund's adjuvant, no bacterial infection was observed in the post-emulsification immunized mice with aluminum hydroxide and H. felis. Inducing type 1 or type 2 immunity is an alternative method of mucosal immunity against Helicobacter pylori. HE staining of mouse sections showed that after immunization with aluminum hydroxide or complete Freund's adjuvant, the mice showed considerable mucosal inflammation in the gastric antrum and fundus mucosa. Degree of inflammation There is no significant difference between the characteristics and degree of inflammation caused by Helicobacter pylori pyrogen immunity and the previously studied cholera toxin adjuvant immunity. The average fundus inflammation of mice immunized with aluminum hydroxide, Freund's complete adjuvant, and Helicobacter pylori antigen was 7.2 + 0.9 and 7.4 + 0.7, respectively. The average gastric antrum inflammation of mice immunized with aluminum hydroxide, Freund’s complete adjuvant, and Helicobacter pylori antigen was 2.0 + 0 and 2.1 + 0.6, respectively. In all cases, mice immunized with Helicobacter pylori antigen were compared with Mice immunized with ovalbumin showed more severe inflammation. This type of gastritis after immunization has also been observed in other experiments. It may be due to secondary immunity to Helicobacter pylori immune memory.

　　Next, we began to study the immune system against Helicobacter pylori after immunization. Complete Freund's adjuvant such as cholera toxin is highly toxic and should not be used in humans. The focus is on the use of aluminum hydroxide adjuvant. The subsequent experiments used the mouse-H.felis model. This microorganism exhibits more obvious inflammation than Helicobacter pylori in mice and causes more serious infections, so it is easy to observe in tissue sections. Aluminum hydroxide AlOH3 or ovalbumin and cat antigen are used to immunize mice homologous uMT (immunoglobulin knockout mice). Whether it can provide protective antibodies after immunization. All eight immunoglobulin knockout mice in the ovalbumin control group were H. After intragastric administration of cats, bacteria are infected and the growth rate of bacteria is high. However, only one immunoglobulin knockout mouse uMT and aluminum hydroxide adjuvant H. felis showed a strong immune defense. In fully immunized B6 mice, these inflammatory responses were not significantly different. After immunization with aluminum hydroxide and H. felis, the inflammation of the antrum was 7.8±0.6, the inflammation of the fundus was 2.38±0.5, and the inflammation of the antrum and the fundus of the stomach after immunization with ovalbumin and aluminum hydroxide were 3.7 + 1.5 and 1.5, respectively + 0.5. This is consistent with the previous results of using oral immunoglobulin to knock out uMT in mice.

　　These experiments show that there is no need for antibody immunization after vaccination. Next, we conducted an experiment to see if we can protect T cells purified from vaccinated mice from adoption. Spleen cells from C57BL/6 mice immunized with H.felis and aluminum hydroxide, ovalbumin and aluminum hydroxide, H.felis and complete Freund's adjuvant, 28 after immunization with ovalbumin and Freund's adjuvant Day, spleen cells and column-purified CD4 T cells from C57BL/6 mice.

　　Discussion: In short, the use of aluminum hydroxide AlOH3 as an adjuvant for systemic immunity has been shown to induce protective mice against Helicobacter pylori and H. ferris, which mediate CD42 cell-mediated immunity. These findings may have a direct impact on the development of human Helicobacter pylori vaccine. First, this report strengthens the concept of system immunity. Approved adjuvants for use in humans may be a viable alternative experiment for inducing mucosal immunity of Helicobacter pylori through oral administration. To further support this, in another series of experiments aimed at studying the effectiveness of parental Helicobacter pylori immunity on newborns, our

　　Success and reproducibility are based on subcutaneous immunization to obtain protective immunity. Guyetal recently reported this view, he introduced a new type of Helicobacter pylori vaccination that violates the principle of long-term mucosal immunity.

　　Secondly, contrary to Gaieta's discovery, Helicobacter pylori requires a type 1 immune response. Our data shows that type 2 immune response is as important as type 1 immune response and may be more effective against Helicobacter pylori infection. Aluminum hydroxide is the only adjuvant available in humans and is an adjuvant that triggers a type 2 immune response. According to some recent reports, it can protect the host from Helicobacter pylori type 2 mucosal immunity. This includes oral cholera toxin and other experimental immune adjuvants that cause type 2 or mixed type 2 and type 1 reactions.