Marygorret Obonyo

Deficiencies of myeloid differentiation factor 88, Toll-like receptor 2 (TLR2), or TLR4 produce specific defects in macrophage cytokine secretion induced by Helicobacter pylori.

ABSTRACT Helicobacter pylori is a gram-negative microaerophilic bacterium that colonizes the gastric mucosa, leading to disease conditions ranging from gastritis to cancer. Toll-like receptors (TLRs) play a central role in innate immunity by their recognition of conserved molecular patterns on bacteria, fungi, and viruses. Upon recognition of microbial components, these TLRs associate with several adaptor molecules, including myeloid differentiation factor 88 (MyD88). To investigate the contribution of the innate immune system to H. pylori infection, bone marrow-derived macrophages from mice deficient in TLR2, TLR4, TLR9, and MyD88 were infected with H. pylori SS1 and SD4 for 24 or 48 h. We demonstrate that MyD88 was essential for H. pylori induction of all cytokines investigated except alpha interferon (IFN-α). The secretion of IFN-α was substantially increased from cells deficient in MyD88. H. pylori induced interleukin-12 (IL-12) and IL-10 through TLR4/MyD88 signaling. In addition, H. pylori induced less IL-6 and IL-1β in TLR2-deleted macrophages, suggesting that the MyD88 pathway activated by TLR2 stimulation is responsible for H. pylori induction of the host proinflammatory response (IL-6 and IL-1β). These observations are important in light of a recent report on IL-6 and IL-1β playing a role in the development of H. pylori-related gastric cancer. In conclusion, our study demonstrates that H. pylori activates TLR2 and TLR4, leading to the secretion of distinct cytokines by macrophages.

Myeloid Differentiation Primary Response Gene 88 Is Required for the Resolution of Otitis Media

BACKGROUND Signaling defects in the Toll-like receptor (TLR) pathway, such as interleukin-1 receptor-associated kinase 4 deficiency, highlight the prominence of TLR signaling in the defense against bacterial disease. Because myeloid differentiation primary response gene 88 (MyD88) can transduce signals from almost all TLRs, we studied its role in otitis media (OM), the most common upper respiratory tract bacterial infectious disease in young children. METHODS The middle ears (MEs) of wild-type (WT) and MyD88(-/-) mice were inoculated with nontypeable Haemophilus influenzae (NTHi). ME infection and inflammation were monitored for 21 days after surgery. Bone marrow-derived macrophages from WT and MyD88(-/-) mice were infected with NTHi in vitro to assess their interaction with bacteria. RESULTS In WT mice, MyD88 expression was detected in the ME stroma at baseline. MyD88(-/-) mice displayed prolonged ME mucosal thickening and delayed recruitment of neutrophils and macrophages. Although WT mice cleared NTHi within 5 days, viable NTHi were isolated for up to 21 days in MyD88(-/-) mice. The interaction between macrophages and NTHi was significantly altered in MyD88(-/-) mice. CONCLUSIONS In this mouse model, MyD88-mediated signaling was important for clearance of infection and resolution of inflammation in acute OM due to NTHi. The role played by innate signaling in children susceptible to chronic or recurrent OM deserves further study.

Role of Gamma Interferon in Helicobacter pylori Induction of Inflammatory Mediators during Murine Infection

ABSTRACT Gamma interferon (IFN-γ) has been proposed to play an important role in Helicobacter-related gastritis. Using the IFN-γ gene knockout (IFN-γ−/−) mouse model and a murine gastric epithelial cell line, GSM06, we demonstrated that Helicobacter pylori maximally induced macrophage inflammatory protein-2 (MIP-2) and inducible nitric oxide synthase (iNOS) mRNA only in wild-type mice. MIP-2 and iNOS mRNA were also induced by H. pylori in GSM06 cells. Induction of cyclooxygenase 2 mRNA through IFN-γ was demonstrated in GSM06 cells. These data indicate that IFN-γ mediates the induction of MIP-2 and iNOS mRNA expression by H. pylori in mice.

Antibacterial activities of liposomal linolenic acids against antibiotic-resistant Helicobacter pylori.

Helicobacter pylori (H. pylori) infection with its vast prevalence is responsible for various gastric diseases including gastritis, peptic ulcers, and gastric malignancy. While effective, current treatment regimens are challenged by a fast-declining eradication rate due to the increasing emergence of H. pylori strains resistant to existing antibiotics. Therefore, there is an urgent need to develop novel antibacterial strategies against H. pylori. In this study, we developed a liposomal nanoformulation of linolenic acid (LipoLLA) and evaluated its bactericidal activity against resistant strains of H. pylori. Using a laboratory strain of H. pylori, we found that LipoLLA was effective in killing both spiral and coccoid forms of the bacteria via disrupting bacterial membranes. Using a metronidazole-resistant strain of H. pylori and seven clinically isolated strains, we further demonstrated that LipoLLA eradicated all strains of the bacteria regardless of their antibiotic resistance status. Furthermore, under our experimental conditions, the bacteria did not develop drug resistance when cultured with LipoLLA at various sub-bactericidal concentrations, whereas they rapidly acquired resistance to both metronidazole and free linolenic acid (LLA). Our findings suggest that LipoLLA is a promising antibacterial nanotherapeutic to treat antibiotic-resistant H. pylori infection.

In vivo treatment of Helicobacter pylori infection with liposomal linolenic acid reduces colonization and ameliorates inflammation

Significance The use of a liposomal formulation of linolenic acid to kill Helicobacter pylori bacteria in the stomach represents a powerful treatment option for H. pylori infection, as well as its associated gastroduodenal diseases. Because the therapeutic agent is a natural compound found in common vegetable oils, such treatment is expected to be cost-effective compared with the existing antibiotic-based anti-H. pylori therapeutics. More important, the excellent antimicrobial efficacy, marked by significantly reduced in vivo bacterial colonization and inflammation, is achieved through rapid fusion of the nanoformulation with bacterial membrane, a mechanism known to overcome bacterial drug resistance. Similar therapeutic approaches can be developed to treat various types other bacterial infections. Helicobacter pylori infection is marked by a vast prevalence and strong association with various gastric diseases, including gastritis, peptic ulcers, and gastric cancer. Because of the rapid emergence of H. pylori strains resistant to existing antibiotics, current treatment regimens show a rapid decline of their eradication rates. Clearly, novel antibacterial strategies against H. pylori are urgently needed. Here, we investigated the in vivo therapeutic potential of liposomal linolenic acid (LipoLLA) for the treatment of H. pylori infection. The LipoLLA formulation with a size of ∼100 nm was prone to fusion with bacterial membrane, thereby directly releasing a high dose of linolenic acids into the bacterial membrane. LipoLLA penetrated the mucus layer of mouse stomach, and a significant portion of the administered LipoLLA was retained in the stomach lining up to 24 h after the oral administration. In vivo tests further confirmed that LipoLLA was able to kill H. pylori and reduce bacterial load in the mouse stomach. LipoLLA treatment was also shown to reduce the levels of proinflammatory cytokines including interleukin 1β, interleukin 6, and tumor necrosis factor alpha, which were otherwise elevated because of the H. pylori infection. Finally, a toxicity test demonstrated excellent biocompatibility of LipoLLA to normal mouse stomach. Collectively, results from this study indicate that LipoLLA is a promising, effective, and safe therapeutic agent for the treatment of H. pylori infection.

High-Fat, High-Cholesterol Diet Increases the Incidence of Gastritis in LDL Receptor–Negative Mice

Transgenic and knockout mice are widely used as models for atherogenesis studies. While developing a Helicobacter infection model in LDL receptor–negative (LDLR−/−) mice, we noticed that mice fed a high-fat, high-cholesterol diet often contracted gastritis independent of infection. To further investigate this finding, we studied 27 male and 18 female LDLR−/− mice fed high-fat, 1% or 1.25% cholesterol diets for 3 to 4 months. The extent of atherosclerosis was morphometrically analyzed in the whole aorta, and the degree of gastric inflammation was scored histologically in hematoxylin-eosin–stained stomach sections. The autoantibody titers to epitopes of oxidized LDL were also measured. Mice fed high-fat, high-cholesterol diets had a significantly higher incidence of gastritis than mice fed normal chow, 62% versus 5%, respectively (P <0.0001). This effect was specific for LDLR−/− mice, because no difference in gastritis was found in wild-type mice fed either diet. Animals with gastritis showed slightly more atherosclerosis than animals without gastritis: 16.3±6.4% versus 12.8±3.4% in males and 9.4±3.5% versus 6.5±3.3% in females. Cholesterol-fed mice also had significantly higher IgG autoantibody titers against modified LDL than normal chow–fed animals, but no difference was seen between the gastritis and nongastritis groups. We conclude that the standard high-fat, high-cholesterol diet commonly used in many murine models to induce atherosclerosis increased the incidence of gastritis significantly in LDLR−/− mice.

Mechanism of antibacterial activity of liposomal linolenic acid against Helicobacter pylori.

Helicobacter pylori infects approximately half of the world population and is a major cause of gastritis, peptic ulcer, and gastric cancer. Moreover, this bacterium has quickly developed resistance to all major antibiotics. Recently, we developed a novel liposomal linolenic acid (LipoLLA) formulation, which showed potent bactericidal activity against several clinical isolated antibiotic-resistant strains of H. pylori including both the spiral and coccoid form. In addition, LipoLLA had superior in vivo efficacy compared to the standard triple therapy. Our data showed that LipoLLA associated with H. pylori cell membrane. Therefore, in this study, we investigated the possible antibacterial mechanism of LipoLLA against H. pylori. The antibacterial activity of LipoLLA (C18:3) was compared to that of liposomal stearic acid (LipoSA, C18:0) and oleic acid (LipoOA, C18:1). LipoLLA showed the most potent bactericidal effect and completely killed H. pylori within 5 min. The permeability of the outer membrane of H. pylori increased when treated with LipoOA and LipoLLA. Moreover, by detecting released adenosine triphosphate (ATP) from bacteria, we found that bacterial plasma membrane of H. pylori treated with LipoLLA exhibited significantly higher permeability than those treated with LipoOA, resulting in bacteria cell death. Furthermore, LipoLLA caused structural changes in the bacterial membrane within 5 min affecting membrane integrity and leading to leakage of cytoplasmic contents, observed by both transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Our findings showing rapid bactericidal effect of LipoLLA suggest it is a very promising new, effective anti-H. pylori agent.

Deficiency of the Myeloid Differentiation Primary Response Molecule MyD88 Leads to an Early and Rapid Development of Helicobacter-Induced Gastric Malignancy

ABSTRACT Approximately 50% of the worlds population is infected with Helicobacter pylori, leading to chronic inflammation, which increases the risk for gastric adenocarcinoma. MyD88 is a key adaptor molecule in inflammatory pathways involved in interleukin 1 (IL-1)/IL-18/Toll-like receptor signaling and has been shown to have divergent effects in carcinogenesis. The role of MyD88 in Helicobacter-induced gastric malignancy is unknown. Using a mouse model of Helicobacter-induced gastric cancer, we assessed the role of MyD88 in cancer development by evaluating gastric histopathology, apoptosis, proliferation, and cytokine expression. Infection of MyD88-deficient (Myd88 −/−) mice with Helicobacter resulted in early and rapid advancement to gastric dysplasia as early as 25 weeks postinfection. The progression of Helicobacter-induced disease to precancerous and cancerous lesions in the absence of MyD88 signaling was accompanied by increased gastric epithelial apoptosis and proliferation. In addition, inflammatory cytokines, including tumor necrosis factor alpha (TNF-α), gamma interferon (IFN-γ), IL-6, and IL-1β were highly expressed in association with the development of gastric dysplasia. These data suggest that MyD88 signaling retards development and progression to cancer during Helicobacter infection. This is the first study to show evidence of MyD88 protection in an infection-driven inflammation-associated cancer model.

Effects of Myeloid Differentiation Primary Response Gene 88 (MyD88) Activation on Helicobacter Infection In Vivo and Induction of a Th17 Response

Background: Helicobacter pylori is a spiral‐shaped Gram‐negative microaerophilic bacterium associated with a number of gastrointestinal disorders, including gastritis, peptic ulcers, and gastric cancer. Several studies have implicated a Th17 response as a key to protective immunity against Helicobacter.

Interactions between Inducible Nitric Oxide and Other Inflammatory Mediators during Helicobacter pylori Infection

Background.  Recent studies in both humans and animal models strongly suggest the contribution of the host immune response to Helicobacter pylori‐related disease. Inducible nitric oxide synthase has been shown to be up‐regulated in the gastric epithelium during H. pylori gastritis, suggesting a role in inflammation.