TRIF-IFN-I pathway in Helicobacter-induced gastric cancer in an accelerated murine disease model and patient biopsies.

Helicobacter pylori (H. pylori) infection is a known cause of many digestive diseases, including gastritis, peptic ulcers, and gastric cancer. However, the underlying mechanisms by which H. pylori infection triggers these disorders are still not clearly understood. Gastric cancer is a slow progressing disease, which makes it difficult to study. We have developed an accelerated disease progression mouse model, which leverages mice deficient in the myeloid differentiation primary response 88 gene (Myd88-/-) infected with Helicobacter felis (H. felis). Using this model and gastric biopsy samples from patients, we report that activation of the Toll/interleukin-1 receptor (TIR)-domain-containing adaptor inducing interferon-ß (TRIF)-type I interferon (IFN-I) signaling pathway promotes Helicobacter-induced disease progression toward severe gastric pathology and gastric cancer development. Further, results implicated downstream targets of this pathway in disease pathogenesis. These findings may facilitate stratification of Helicobacter-infected patients and thus enable treatment prioritization of patients.

Humanized Anti-Carcinoembryonic Antigen Antibodies Brightly Target and Label Gastric Cancer in Orthotopic Mouse Models.

INTRODUCTION: Gastric cancer poses a major therapeutic challenge. Improved visualization of tumor margins at the time of gastrectomy with fluorescent tumor-specific antibodies could improve outcomes. The present report demonstrates the potential of targeting gastric cancer with a humanized anti-carcinoembryonic antigen (CEA) antibody in orthotopic mouse models. METHODS: MKN45 cells were injected subcutaneously into nude mice to establish xenograft models. Tumor fragments collected from subcutaneous models were then implanted into the greater curvature of the stomach to establish orthotopic models. For tumor labeling, a humanized anti-CEA antibody (M5A) and IgG as a control, were conjugated with the near-infrared dye IRDye800CW. Time (24-72 h) and dose (50-100 µg) response curves were performed in subcutaneous models. Orthotopic models received 50 µg of M5A-IR800 or 50 µg IgG-IR800 as a control and were imaged after 72 h. Fluorescence imaging was performed on the mice using the LI-COR Pearl Imaging System. RESULTS: In subcutaneous models, tumor to background ratios (TBRs) reached 8.85 at 72 h. Median TBRs of orthotopic model primary tumors were 6.25 (interquartile range [IQR] 6.03-7.12) for M5A-IR800 compared to 0.42 (IQR 0.38-0.54) for control. Abdominal wall metastasis median TBRs were 13.52 (IQR 12.79-13.76) for M5A-IR800 and 3.19 (IQR 2.65-3.73) for the control. Immunohistochemistry confirmed CEA expression within tumors. CONCLUSIONS: Humanized anti-CEA antibodies conjugated to near-infrared dyes provide specific labeling of gastric cancers in mouse models. Orthotopic models demonstrated bright and specific labeling with TBRs greater than ten times that of control. This tumor-specific fluorescent antibody is a promising potential clinical tool for improving visualization of gastric cancer margins at time of surgical resection.

Activation of the TRIF pathway and downstream targets results in the development of precancerous lesions during infection with Helicobacter.

Helicobacter pylori ( H. pylori) infection is an established cause of many digestive diseases, including gastritis, peptic ulcers, and gastric cancer. However, the mechanism by which infection with H. pylori causes these disorders is still not clearly understood. This is due to insufficient knowledge of pathways that promote H. pylori -induced disease progression. We have established a Helicobacter -induced accelerated disease progression mouse model, which involves infecting mice deficient in the myeloid differentiation primary response 88 gene ( Myd88 -/- ) with H. felis . Using this model, we report here that that progression of H. felis -induced inflammation to high-grade dysplasia was associated with activation of type I interferon (IFN-I) signaling pathway and upregulation of related downstream target genes, IFN-stimulated genes (ISGs). These observations were further corroborated by the enrichment of ISRE motifs in the promoters of upregulated genes. Further we showed that H. felis -induced inflammation in mice deficient in Toll/interleukin-1 receptor (TIR)-domain-containing adaptor inducing interferon-ß (TRIF, Trif Lps 2 ) did not progress to severe gastric pathology, indicating a role of the TRIF signaling pathway in disease pathogenesis and progression. Indeed, survival analysis in gastric biopsy samples from gastric cancer patients illustrated that high expression of Trif was significantly associated with poor survival in gastric cancer.

A Bioluminescence-Based Drug Screen Identifies Activities of Fexinidazole and Its Metabolites against Helicobacter pylori.

Helicobacter pylori is responsible for a wide range of gastric diseases, including gastric cancer and gastritis. With half of the world's population infected by H. pylori and the current standard of care associated with suboptimal outcomes, a search for more effective drugs is critical. To facilitate drug screening for H. pylori, we developed a microtiter plate-based compound screening method that is faster and can screen multiple compounds. We identified activities of fexinidazole and its sulfoxide and sulfone metabolites against H. pylori. Both fexinidazole and its metabolites exhibited equipotency against SS1, 60190, and G27 strains, which were about 3-6-fold more potent than the currently used metronidazole. We also determined the minimal inhibitory concentration (MIC) of metronidazole, fexinidazole, and its metabolites against these strains by a traditional agar plate-based method. While MIC values of fexinidazole and metronidazole were similar against all the strains, both sulfoxide and sulfone showed lower MIC values than metronidazole against SS1 and 60190. Given the recent FDA approval of fexinidazole, our data on the in vitro antibacterial activities of fexinidazole and its metabolites support further evaluation of this drug with the goal of producing an alternative nitro-based antimicrobial with good safety profiles for the treatment of H. pylori infection.

Genomically Silent Refractory Gastric Cancer in a Young Patient Exhibits Overexpression of CXCL5.

Gastric cancer is the third leading cause of cancer-related deaths, with more than one million new cases and approximately 841,000 deaths annually worldwide. We report a case of a young patient (25 years old) with an aggressive form of gastric cancer. The patient had previously been treated for Helicobacter pylori (H. pylori), which is a main risk factor for developing gastric cancer. Genetic testing showed an E-cadherin (CDH1) germline mutation of unknown significance. After eight cycles of chemotherapy, a positron emission tomography (PET) scan showed disease progression with an enlarging hypermetabolic right adnexal mass suspicious for metastatic disease. Tumor pathology demonstrated invasive and poorly differentiated gastric carcinoma. The analysis of the tumor biopsy indicated the very high expression of a chemokine, C-X-C motif chemokine 5 (CXCL5). The combination of H. pylori infection with an existence of a rare CDH1 mutation could have contributed to this aggressive gastric cancer.

Early detection of tumor cells in bone marrow and peripheral blood in a fast­progressing gastric cancer model.

Helicobacter pylori (H. pylori) infection is a major risk factor for the development of gastric cancer. The authors previously demonstrated that in mice deficient in myeloid differentiation primary response 88 (Myd88­/­), infection with Helicobacter felis (H. felis) a close relative of H. pylori, subsequently rapidly progressed to neoplasia. The present study examined circulating tumor cells (CTCs) by measuring the expression of cytokeratins, epithelial­to­mesenchymal transition (EMT)­related markers and cancer stem cell (CSC) markers in bone marrow and peripheral blood from Myd88­/­ and wild­type (WT) mice. Cytokeratins CK8/18 were detected as early as 4 months post­infection in Myd88­/­ mice. By contrast, cytokeratins were not detected in WT mice even after 7 months post­infection. The expression of Mucin­1 (MUC1) was observed in both bone marrow and peripheral blood at different time points, suggesting its role in gastric cancer metastasis. Snail, Twist and ZEB were expressed at different levels in bone marrow and peripheral blood. The expression of these EMT­related markers suggests the manifestation of cancer metastasis in the early stages of disease development. LGR5, CD44 and CD133 were the most prominent CSC markers detected. The detection of CSC and EMT markers along with cytokeratins does reinforce their use as biomarkers for gastric cancer metastasis. This early detection of markers suggests that CTCs leave primary site even before cancer is well established. Thus, cytokeratins, EMT, and CSCs could be used as biomarkers to detect aggressive forms of gastric cancers. This information may prove to be of significance in stratifying patients for treatment prior to the onset of severe disease­related characteristics.

Microbiome Signatures in a Fast- and Slow-Progressing Gastric Cancer Murine Model and Their Contribution to Gastric Carcinogenesis.

Gastric cancer is the third most common cause of death from cancer in the world and infection with Helicobacter pylori (H. pylori) is the main cause of gastric cancer. In addition to Helicobacter infection, the overall stomach microbiota has recently emerged as a potential factor in gastric cancer progression. Previously we had established that mice deficient in myeloid differentiation primary response gene 88 (MyD88, Myd88-/- ) rapidly progressed to neoplasia when infected with H. felis. Thus, in order to assess the role of the microbiota in this fast-progressing gastric cancer model we investigated changes of the gastric microbiome in mice with different genotypic backgrounds: wild type (WT), MyD88-deficient (Myd88-/- ), mice deficient in the Toll/interleukin-1 receptor (TIR) domain-containing adaptor-inducing interferon-ß (TRIF, Trif Lps2), and MyD88- and TRIF-deficient (Myd88-/- /Trif Lps2, double knockout (DKO)) mice. We compared changes in alpha diversity, beta diversity, relative abundance, and log-fold differential of relative abundance ratios in uninfected and Helicobacter infected mice and studied their correlations with disease progression to gastric cancer in situ. We observed an overall reduction in microbial diversity post-infection with H. felis across all genotypes. Campylobacterales were observed in all infected mice, with marked reduction in abundance at 3 and 6 months in Myd88-/- mice. A sharp increase in Lactobacillales in infected Myd88-/- and DKO mice at 3 and 6 months was observed as compared to Trif Lps2 and WT mice, hinting at a possible role of these bacteria in gastric cancer progression. This was further reinforced upon comparison of Lactobacillales log-fold differentials with histological data, indicating that Lactobacillales are closely associated with Helicobacter infection and gastric cancer progression. Our study suggests that differences in genotypes could influence the stomach microbiome and make it more susceptible to the development of gastric cancer upon Helicobacter infection. Additionally, increase in Lactobacillales could contribute to faster development of gastric cancer and might serve as a potential biomarker for the fast progressing form of gastric cancer.

Inhibition of Pathogen Adhesion by Bacterial Outer Membrane-Coated Nanoparticles.

Anti-adhesion therapies interfere with the bacterial adhesion to the host and thus avoid direct disruption of bacterial cycles for killing, which may alleviate resistance development. Herein, an anti-adhesion nanomedicine platform is made by wrapping synthetic polymeric cores with bacterial outer membranes. The resulting bacterium-mimicking nanoparticles (denoted "OM-NPs") compete with source bacteria for binding to the host. The "top-down" fabrication of OM-NPs avoids the identification of the adhesins and bypasses the design of agonists targeting these adhesins. In this study, OM-NPs are made with the membrane of Helicobacter pylori and shown to bind with gastric epithelial cells (AGS cells). Treatment of AGS cells with OM-NPs reduces H. pylori adhesion and such anti-adhesion efficacy is dependent on OM-NP concentration and its dosing sequence.

Increased LIGHT expression and activation of non-canonical NF-κB are observed in gastric lesions of MyD88-deficient mice upon Helicobacter felis infection.

Helicobacter pylori infection induces a number of pro-inflammatory signaling pathways contributing to gastric inflammation and carcinogenesis. Among those, NF-κB signaling plays a pivotal role during infection and malignant transformation of the gastric epithelium. However, deficiency of the adaptor molecule myeloid differentiation primary response 88 (MyD88), which signals through NF-κB, led to an accelerated development of gastric pathology upon H. felis infection, but the mechanisms leading to this phenotype remained elusive. Non-canonical NF-κB signaling was shown to aggravate H. pylori-induced gastric inflammation via activation of the lymphotoxin ß receptor (LTßR). In the present study, we explored whether the exacerbated pathology observed in MyD88-deficient (Myd88-/-) mice was associated with aberrant activation of non-canonical NF-κB. Our results indicate that, in the absence of MyD88, H. felis infection enhances the activation of non-canonical NF-κB that is associated with increase in Cxcl9 and Icam1 gene expression and CD3+ lymphocyte recruitment. In addition, activation of signal transducer and activator of transcription 3 (STAT3) signaling was higher in Myd88-/- compared to wild type (WT) mice, indicating a link between MyD88 deficiency and STAT3 activation in response to H. felis infection. Thereby, MyD88 deficiency results in accelerated and aggravated gastric pathology induced by Helicobacter through activation of non-canonical NF-κB.

Coating nanoparticles with gastric epithelial cell membrane for targeted antibiotic delivery against Helicobacter pylori infection.

Inspired by the natural pathogen-host interactions and adhesion, this study reports on the development of a novel targeted nanotherapeutics for the treatment of Helicobacter pylori (H. pylori) infection. Specifically, plasma membranes of gastric epithelial cells (e.g. AGS cells) are collected and coated onto antibiotic-loaded polymeric cores, the resulting biomimetic nanoparticles (denoted AGS-NPs) bear the same surface antigens as the source AGS cells and thus have inherent adhesion to H. pylori bacteria. When incubated with H. pylori bacteria in vitro, the AGS-NPs preferentially accumulate on the bacterial surfaces. Using clarithromycin (CLR) as a model antibiotic and a mouse model of H. pylori infection, the CLR-loaded AGS-NPs demonstrate superior therapeutic efficacy as compared the free drug counterpart as well as non-targeted nanoparticle control group. Overall, this work illustrates the promise and strength of using natural host cell membranes to functionalize drug nanocarriers for targeted drug delivery to pathogens that colonize on the host cells. As host-pathogen adhesion represents a common biological event for various types of pathogenic bacteria, the bioinspired nanotherapeutic strategy reported here represents a versatile delivery platform that may be applied to treat numerous infectious diseases.

Comparison of lipopolysaccharides composition of two different strains of Helicobacter pylori.

BACKGROUND: Helicobacter pylori (H. pylori) is a Gram-negative, microaerophilic bacterium that is recognized as a major cause of chronic gastritis, peptic ulcers, and gastric cancer. Comparable to other Gram-negative bacteria, lipopolysaccharides (LPS) are an important cellular component of the outer membrane of H. pylori. The LPS of this organism plays a key role in its colonization and persistence in the stomach. In addition, H. pylori LPS modulates pathogen-induced host inflammatory responses resulting in chronic inflammation within the gastrointestinal tract. Very little is known about the comparative LPS compositions of different strains of H. pylori with varied degree of virulence in human. Therefore, LPS was analyzed from two strains of H. pylori with differing potency in inducing inflammatory responses (SS1 and G27). LPS were extracted from aqueous and phenol layer of hot-phenol water extraction method and subjected for composition analysis by gas chromatography - mass spectrometry (GC-MS) to sugar and fatty acid compositions. RESULTS: The major difference between the two strains of H. pylori is the presence of Rhamnose, Fucose and GalNAc in the SS1 strain, which was either not found or with low abundance in the G27 strain. On the other hand, high amount of Mannose was present in G27 in comparison to SS1. Fatty acid composition of lipid-A portion also showed considerable amount of differences between the two strains, phenol layer of SS1 had enhanced amount of 3 hydroxy decanoic acid (3-OH-C10:0) and 3-hydroxy dodecanoic acid (3-OH-C12:0) which were not present in G27, whereas myristic acid (C14:0) was present in G27 in relatively high amount. CONCLUSION: The composition analysis of H. pylori LPS, revealed differences in sugars and fatty acids composition between a mouse adapted strain SS1 and G27. This knowledge provides a novel way to dissect out their importance in host-pathogen interaction in further studies.

Author correction: Micromotor-enabled active drug delivery for in vivo treatment of stomach infection.

Marygorret Obonyo, who provided the H. pylori SS1 strain for this work and participated in the design of H. pylori infection studies, was inadvertently omitted from the author list. This has now been corrected in both the PDF and HTML versions of the Article.

Micromotor-enabled active drug delivery for in vivo treatment of stomach infection.

Advances in bioinspired design principles and nanomaterials have led to tremendous progress in autonomously moving synthetic nano/micromotors with diverse functionalities in different environments. However, a significant gap remains in moving nano/micromotors from test tubes to living organisms for treating diseases with high efficacy. Here we present the first, to our knowledge, in vivo therapeutic micromotors application for active drug delivery to treat gastric bacterial infection in a mouse model using clarithromycin as a model antibiotic and Helicobacter pylori infection as a model disease. The propulsion of drug-loaded magnesium micromotors in gastric media enables effective antibiotic delivery, leading to significant bacteria burden reduction in the mouse stomach compared with passive drug carriers, with no apparent toxicity. Moreover, while the drug-loaded micromotors reach similar therapeutic efficacy as the positive control of free drug plus proton pump inhibitor, the micromotors can function without proton pump inhibitors because of their built-in proton depletion function associated with their locomotion.Nano- and micromotors have been demonstrated in vitro for a range of applications. Here the authors demonstrate the in-vivo therapeutic use of micromotors to treat H. pylori infection.

Effect of myeloid differentiation primary response gene 88 on expression profiles of genes during the development and progression of Helicobacter-induced gastric cancer.

BACKGROUND: Gastric cancer is one of the most common and lethal type of cancer worldwide. Infection with Helicobacter pylori (H. pylori) is recognized as the major cause of gastric cancer. However, it remains unclear the mechanism by which Helicobacter infection leads to gastric cancer. Furthermore, the underlying molecular events involved during the progression of Helicobacter infection to gastric malignancy are not well understood. In previous studies, we demonstrated that that H. felis-infected Myd88 -/- mice exhibited dramatic pathology and an accelerated progression to gastric dysplasia; however, the MyD88 downstream gene targets responsible for this pathology have not been described. This study was designed to identify MyD88-dependent genes involved in the progression towards gastric cancer during the course of Helicobacter infection. METHODS: Wild type (WT) and Myd88 deficient mice (Myd88 -/-) were infected with H. felis for 25 and 47 weeks and global transcriptome analysis performed on gastric tissue using MouseWG-6 v2 expression BeadChips microarrays. Function and pathway enrichment analyses of statistically significant, differential expressed genes (p < 0.05) were performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) online tools. RESULTS: Helicobacter infection affected the transcriptional profile of more genes in Myd88 -/- mice compared to WT mice. Infection of Myd88 -/- mice resulted in the differential expression of 1,989 genes at 25 weeks (1031 up and 958 downregulated). At 47 weeks post-H.felis infection, 2,162 (1140 up and 1022 downregulated) were differentially expressed. The most significant differentially upregulated gene during Helicobacter infection in Myd88 -/- mice was chitinase-like 4 (chil4), which is involved in tissue remodeling and wound healing. Other highly upregulated genes in H. felis-infected Myd88 -/- mice included, Indoleamine 2,3-Dioxygenase 1 (Ido1), Guanylate binding protein 2 (Gbp2), ubiquitin D (Ubd), ß 2 -Microglobulin (B2m), CD74 antigen (Cd74), which have been reported to promote cancer progression by enhancing angiogenesis, proliferation, migration, metastasis, invasion, and tumorigenecity. For downregulated genes, the highly expressed genes included, ATPase H+/K+ transporting, alpha subunit (Atp4a), Atp4b, Mucin 5 AC (Muc5ac), Apolipoprotein A-1 (Apoa1), and gastric intrinsic factor (Gif), whose optimal function is important in maintaining gastric hemostasis and lower expression has been associated with increased risk of gastric carcinogenesis. CONCLUSIONS: These results provide a global transcriptional gene profile during the development and progression of Helicobacter-induced gastric cancer. The data show that our mouse model system is useful for identifying genes involved in gastric cancer progression.

Helicobacter pylori modulates host cell survival regulation through the serine-threonine kinase, 3-phosphoinositide dependent kinase 1 (PDK-1).

BACKGROUND: Helicobacter pylori (H. pylori) infection affects cell survival signaling pathways including cell apoptosis and proliferation, which are considered risk factors for the development of gastric cancer when unregulated. In the present study, we investigated the effect of H. pylori infection on the phosphorylation state of 3-phosphoinositide-dependent kinase-1 (PDK-1), a master kinase that regulates phosphorylation of Akt (also known as protein kinase B, PKB) and cell survival. METHODS: The activity of PDK-1 was examined in human gastric epithelial cells incubated in the presence or absence of different H. pylori strains. In addition, the role of H. pylori type IV secretion system and the mechanism of H. pylori effect on PDK-1 activity was examined. RESULTS: In the presence of H. pylori, phosphorylation of the activation loop (serine 241) PDK-1 was rapidly lost suggesting that dephosphorylation of PDK-1 is a target for H. pylori to modulate cell survival. The extent of dephosphorylation was strain dependent with H. pylori 60190 being the most effective. H. pylori infection of gastric epithelial cells resulted in altered phosphorylation and degradation of Akt, suggesting that PDK-1 dephosphorylation affects cell survival pathways and thereby may contribute to disease pathogenesis. CONCLUSION: We propose that dephosphorylation of PDK-1 and the resulting changes to Akt phosphorylation is one of the mechanisms by which infection with H. pylori alter the balance between apoptosis and cell proliferation and identify a host molecular mechanism regulated by H. pylori that ultimately contributes to carcinogenesis. Our studies therefore provide insights into one of the mechanisms by which H. pylori infection contributes to gastric cancer by regulating the activity of a cell survival signaling pathway.

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.

Phospholipase A2-responsive antibiotic delivery via nanoparticle-stabilized liposomes for the treatment of bacterial infection.

Adsorbing small charged nanoparticles onto liposome surfaces to stabilize them against fusion and payload leakage has resulted in a new class of liposomes capable of environment-responsive drug delivery. Herein, we engineered a liposome formulation with a lipid composition sensitive to bacterium-secreted phospholipase A2 (PLA2) and adsorbed chitosan-modified gold nanoparticles (AuChi) onto the liposome surface. The resulting AuChi-stabilized liposomes (AuChi-liposomes) showed prohibited fusion activity and negligible drug leakage. However, upon exposure to either purified PLA2 enzyme or PLA2 secreted by Helicobacter pylori (H. pylori) bacteria in culture, AuChi-liposomes rapidly released the encapsulated payloads and such responsive release was retarded by adding quinacrine dihydrochloride, a PLA2 inhibitor. When loaded with doxycycline, AuChi-liposomes effectively inhibited H. pylori growth. Overall, the AuChi-liposomes allowed for smart "on-demand" antibitoic delivery: the more enzymes or bacteria present at the infection site, the more drug will be released to treat the infection. Given the strong association of PLA2 with a diverse range of diseases, the present liposomal delivery technique holds broad application potential for tissue microenvironment-responsive drug delivery.

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

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 1ß, 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.

Deficiency of the myeloid differentiation primary response molecule MyD88 leads to an early and rapid development of Helicobacter-induced gastric malignancy.

Approximately 50% of the world's 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-1ß 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.

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.