Notch-RBP-J signaling regulates the transcription factor IRF8 to promote inflammatory macrophage polarization.

Emerging concepts suggest that the functional phenotype of macrophages is regulated by transcription factors that define alternative activation states. We found that RBP-J, the main nuclear transducer of signaling via Notch receptors, augmented Toll-like receptor 4 (TLR4)-induced expression of key mediators of classically activated M1 macrophages and thus of innate immune responses to Listeria monocytogenes. Notch-RBP-J signaling controlled expression of the transcription factor IRF8 that induced downstream M1 macrophage-associated genes. RBP-J promoted the synthesis of IRF8 protein by selectively augmenting kinase IRAK2-dependent signaling via TLR4 to the kinase MNK1 and downstream translation-initiation control through eIF4E. Our results define a signaling network in which signaling via Notch-RBP-J and TLRs is integrated at the level of synthesis of IRF8 protein and identify a mechanism by which heterologous signaling pathways can regulate the TLR-induced inflammatory polarization of macrophages.

Empirical rescue treatment of Helicobacter pylori infection in third and subsequent lines: 8-year experience in 2144 patients from the European Registry on H. pylori management (Hp-EuReg).

OBJECTIVE: To evaluate the use, effectiveness and safety of Helicobacter pylori empirical rescue therapy in third and subsequent treatment lines in Europe. DESIGN: International, prospective, non-interventional registry of the clinical practice of European gastroenterologists. Data were collected and quality reviewed until October 2021 at Asociación Española de Gastroenterología-Research Electronic Data Capture. All cases with three or more empirical eradication attempts were assessed for effectiveness by modified intention-to-treat and per-protocol analysis. RESULTS: Overall, 2144 treatments were included: 1519, 439, 145 and 41 cases from third, fourth, fifth and sixth treatment lines, respectively. Sixty different therapies were used; the 15 most frequently prescribed encompassed >90% of cases. Overall effectiveness remained <90% in all therapies. Optimised treatments achieved a higher eradication rate than non-optimised (78% vs 67%, p<0.0001). From 2017 to 2021, only 44% of treatments other than 10-day single-capsule therapy used high proton-pump inhibitor doses and lasted ≥14 days. Quadruple therapy containing metronidazole, tetracycline and bismuth achieved optimal eradication rates only when prescribed as third-line treatment, either as 10-day single-capsule therapy (87%) or as 14-day traditional therapy with tetracycline hydrochloride (95%). Triple amoxicillin-levofloxacin therapy achieved 90% effectiveness in Eastern Europe only or when optimised. The overall incidence of adverse events was 31%. CONCLUSION: Empirical rescue treatment in third and subsequent lines achieved suboptimal effectiveness in most European regions. Only quadruple bismuth-metronidazole-tetracycline (10-day single-capsule or 14-day traditional scheme) and triple amoxicillin-levofloxacin therapies reached acceptable outcomes in some settings. Compliance with empirical therapy optimisation principles is still poor 5 years after clinical practice guidelines update. TRIAL REGISTRATION NUMBER: NCT02328131.

Empirical Second-Line Therapy in 5000 Patients of the European Registry on Helicobacter pylori Management (Hp-EuReg).

BACKGROUND & AIMS: After a first Helicobacter pylori eradication attempt, approximately 20% of patients will remain infected. The aim of the current study was to assess the effectiveness and safety of second-line empiric treatment in Europe. METHODS: This international, multicenter, prospective, non-interventional registry aimed to evaluate the decisions and outcomes of H pylori management by European gastroenterologists. All infected adult cases with a previous eradication treatment attempt were registered with the Spanish Association of Gastroenterology-Research Electronic Data Capture until February 2021. Patients allergic to penicillin and those who received susceptibility-guided therapy were excluded. Data monitoring was performed to ensure data quality. RESULTS: Overall, 5055 patients received empiric second-line treatment. Triple therapy with amoxicillin and levofloxacin was prescribed most commonly (33%). The overall effectiveness was 82% by modified intention-to-treat analysis and 83% in the per-protocol population. After failure of first-line clarithromycin-containing treatment, optimal eradication (>90%) was obtained with moxifloxacin-containing triple therapy or levofloxacin-containing quadruple therapy (with bismuth). In patients receiving triple therapy containing levofloxacin or moxifloxacin, and levofloxacin-bismuth quadruple treatment, cure rates were optimized with 14-day regimens using high doses of proton pump inhibitors. However, 3-in-1 single capsule or levofloxacin-bismuth quadruple therapy produced reliable eradication rates regardless of proton pump inhibitor dose, duration of therapy, or previous first-line treatment. The overall incidence of adverse events was 28%, and most (85%) were mild. Three patients developed serious adverse events (0.3%) requiring hospitalization. CONCLUSIONS: Empiric second-line regimens including 14-day quinolone triple therapies, 14-day levofloxacin-bismuth quadruple therapy, 14-day tetracycline-bismuth classic quadruple therapy, and 10-day bismuth quadruple therapy (as a single capsule) provided optimal effectiveness. However, many other second-line treatments evaluated reported low eradication rates. ClincialTrials.gov number: NCT02328131.

Investigation of Clarithromycin Resistance-Associated Mutations and Virulence Genotypes of Helicobacter pylori Isolated from Iranian Population: A Cross-Sectional Study.

Antibiotic resistance has brought into question the efficiency of clarithromycin which is a vital component of eradication therapy for Helicobacter pylori infection. The point mutations within the 23S rRNA sequence of H. pylori isolates which contribute to clarithromycin resistance have yet to be fully characterized. This study was aimed to detect clarithromycin resistance-associated mutations and assess the prevalence of key virulence factors of H. pylori among Iranian patients. Amplification of 16S rRNA and glmM genes were done to identify H. pylori. Minimal inhibitory concentration (MIC) of clarithromycin in 82 H. pylori clinical isolates was determined by agar dilution method. Subsequently, various virulence markers including cagA, vacA, sabA, babA, and dupA of H. pylori were identified by PCR. PCR-sequencing was applied to detect point mutations in the 23S rRNA gene. Based on MIC values, 43.9% of H. pylori isolates showed resistance to clarithromycin. The babA and cagA genes were detected in 92.7% and 82.9% of isolates, assigned to be higher than other virulence factors. No significant relationship was found between the H. pylori virulence genotypes and clarithromycin susceptibility (P > 0.05). Analyzing the 23S rRNA sequences revealed A2143G (4/48, 8.3%) and A2142G (3/48, 6.2%) as the most prevalent mutations in clarithromycin-resistant isolates. Additionally, several novel mutations including G2220T, C2248T, A2624C, G2287A, T2188C, G2710C, C2248T, G2269A, and G2224T were also detected among either resistant or susceptible isolates. Our findings revealed the presence of several point mutations in the 23S rRNA gene of H. pylori isolates which may be associated with resistance to clarithromycin.

Helicobacter pylori resistance to current therapies.

PURPOSE OF REVIEW: Helicobacter pylori eradication rates have fallen in recent years, mainly because of the emergence of antibiotic-resistant infections. Indeed the WHO has recently designated clarithromycin-resistant H. pylori infection a high priority for antibiotic resistance research and development. This review aims to discuss the most up-to-date information on the methods to detect H. pylori antibiotic resistance, the recent data on resistance rates, and the most appropriate treatment strategies to overcome antibiotic resistance. RECENT FINDINGS: There has been active research into the development and assessment of genotypic diagnostic assays for both the invasive and noninvasive detection of antibiotic-resistant infection. There are regional variations in the prevalence of H. pylori antibiotic resistance. Primary resistance rates in general are on the rise and high rates of clarithromycin resistance (>15%) have been reported in many parts of the world. SUMMARY: Optimizing antimicrobial susceptibility testing by both invasive and noninvasive means is crucial to accurately evaluate resistance rates for the optimization of both regional and personalized H. pylori treatment strategies.

A GWAS on Helicobacter pylori strains points to genetic variants associated with gastric cancer risk.

BACKGROUND: Helicobacter pylori are stomach-dwelling bacteria that are present in about 50% of the global population. Infection is asymptomatic in most cases, but it has been associated with gastritis, gastric ulcers and gastric cancer. Epidemiological evidence shows that progression to cancer depends upon the host and pathogen factors, but questions remain about why cancer phenotypes develop in a minority of infected people. Here, we use comparative genomics approaches to understand how genetic variation amongst bacterial strains influences disease progression. RESULTS: We performed a genome-wide association study (GWAS) on 173 H. pylori isolates from the European population (hpEurope) with known disease aetiology, including 49 from individuals with gastric cancer. We identified SNPs and genes that differed in frequency between isolates from patients with gastric cancer and those with gastritis. The gastric cancer phenotype was associated with the presence of babA and genes in the cag pathogenicity island, one of the major virulence determinants of H. pylori, as well as non-synonymous variations in several less well-studied genes. We devised a simple risk score based on the risk level of associated elements present, which has the potential to identify strains that are likely to cause cancer but will require refinement and validation. CONCLUSION: There are a number of challenges to applying GWAS to bacterial infections, including the difficulty of obtaining matched controls, multiple strain colonization and the possibility that causative strains may not be present when disease is detected. Our results demonstrate that bacterial factors have a sufficiently strong influence on disease progression that even a small-scale GWAS can identify them. Therefore, H. pylori GWAS can elucidate mechanistic pathways to disease and guide clinical treatment options, including for asymptomatic carriers.

RBP-J-Regulated miR-182 Promotes TNF-α-Induced Osteoclastogenesis.

Increased osteoclastogenesis is responsible for osteolysis, which is a severe consequence of inflammatory diseases associated with bone destruction, such as rheumatoid arthritis and periodontitis. The mechanisms that limit osteoclastogenesis under inflammatory conditions are largely unknown. We previously identified transcription factor RBP-J as a key negative regulator that restrains TNF-α-induced osteoclastogenesis and inflammatory bone resorption. In this study, we tested whether RBP-J suppresses inflammatory osteoclastogenesis by regulating the expression of microRNAs (miRNAs) important for this process. Using high-throughput sequencing of miRNAs, we obtained the first, to our knowledge, genome-wide profile of miRNA expression induced by TNF-α in mouse bone marrow-derived macrophages/osteoclast precursors during inflammatory osteoclastogenesis. Furthermore, we identified miR-182 as a novel miRNA that promotes inflammatory osteoclastogenesis driven by TNF-α and whose expression is suppressed by RBP-J. Downregulation of miR-182 dramatically suppressed the enhanced osteoclastogenesis program induced by TNF-α in RBP-J-deficient cells. Complementary loss- and gain-of-function approaches showed that miR-182 is a positive regulator of osteoclastogenic transcription factors NFATc1 and B lymphocyte-induced maturation protein-1. Moreover, we identified that direct miR-182 targets, Foxo3 and Maml1, play important inhibitory roles in TNF-α-mediated osteoclastogenesis. Thus, RBP-J-regulated miR-182 promotes TNF-α-induced osteoclastogenesis via inhibition of Foxo3 and Maml1. Suppression of miR-182 by RBP-J serves as an important mechanism that restrains TNF-α-induced osteoclastogenesis. Our results provide a novel miRNA-mediated mechanism by which RBP-J inhibits osteoclastogenesis and suggest that targeting of the newly described RBP-J-miR-182-Foxo3/Maml1 axis may represent an effective therapeutic approach to suppress inflammatory osteoclastogenesis and bone resorption.

LFA-1/ICAM-1 Ligation in Human T Cells Promotes Th1 Polarization through a GSK3ß Signaling-Dependent Notch Pathway.

In this study, we report that the integrin LFA-1 cross-linking with its ligand ICAM-1 in human PBMCs or CD4(+) T cells promotes Th1 polarization by upregulating IFN-γ secretion and T-bet expression. LFA-1 stimulation in PBMCs, CD4(+) T cells, or the T cell line HuT78 activates the Notch pathway by nuclear translocation of cleaved Notch1 intracellular domain (NICD) and upregulation of target molecules Hey1 and Hes1. Blocking LFA-1 by a neutralizing Ab or specific inhibition of Notch1 by a γ-secretase inhibitor substantially inhibits LFA-1/ICAM-1-mediated activation of Notch signaling. We further demonstrate that the Notch pathway activation is dependent on LFA-1/ICAM-1-induced inactivation of glycogen synthase kinase 3ß (GSK3ß), which is mediated via Akt and ERK. Furthermore, in silico analysis in combination with coimmunoprecipitation assays show an interaction between NICD and GSK3ß. Thus, there exists a molecular cross-talk between LFA-1 and Notch1 through the Akt/ERK-GSK3ß signaling axis that ultimately enhances T cell differentiation toward Th1. Although clinical use of LFA-1 antagonists is limited by toxicity related to immunosuppression, these findings support the concept that Notch inhibitors could be attractive for prevention or treatment of Th1-related immunologic disorders and have implications at the level of local inflammatory responses.

Differential modulation of Helicobacter pylori lipopolysaccharide-mediated TLR2 signaling by individual Pellino proteins.

BACKGROUND: Eradication rates for current H. pylori therapies have fallen in recent years, in line with the emergence of antibiotic resistant infections. The development of therapeutic alternatives to antibiotics, such as immunomodulatory therapy and vaccines, requires a more lucid understanding of host-pathogen interactions, including the relationships between the organism and the innate immune response. Pellino proteins are emerging as key regulators of immune signaling, including the Toll-like receptor pathways known to be regulated by H. pylori. The aim of this study was to characterize the role of Pellino proteins in the innate immune response to H. pylori lipopolysaccharide. MATERIALS AND METHODS: Gain-of-function and loss-of-function approaches were utilized to elucidate the role of individual Pellino proteins in the Toll-like receptor 2-mediated response to H. pylori LPS by monitoring NF-ĸB activation and the induction of proinflammatory chemokines. Expression of Pellino family members was investigated in gastric epithelial cells and gastric tissue biopsy material. RESULTS: Pellino1 and Pellino2 positively regulated Toll-like receptor 2-driven responses to H. pylori LPS, whereas Pellino3 exerted a negative modulatory role. Expression of Pellino1 was significantly higher than Pellino3 in gastric epithelial cells and gastric tissue. Furthermore, Pellino1 expression was further augmented in gastric epithelial cells in response to infection with H. pylori or stimulation with H. pylori LPS. CONCLUSIONS: The combination of low Pellino3 levels together with high and inducible Pellino1 expression may be an important determinant of the degree of inflammation triggered upon Toll-like receptor 2 engagement by H. pylori and/or its components, contributing to H. pylori-associated pathogenesis by directing the incoming signal toward an NF-kB-mediated proinflammatory response.

Repression of the proapoptotic cellular BIK/NBK gene by Epstein-Barr virus antagonizes transforming growth factor ß1-induced B-cell apoptosis.

UNLABELLED: The Epstein-Barr virus (EBV) establishes a lifelong latent infection in humans. EBV infection of primary B cells causes cell activation and proliferation, a process driven by the viral latency III gene expression program, which includes EBV nuclear proteins (EBNAs), latent membrane proteins, and untranslated RNAs, including microRNAs. Some latently infected cells enter the long-lived memory B-cell compartment and express only EBNA1 transiently (Lat I) or no EBV protein at all (Lat 0). Targeting the molecular machinery that controls B-cell fate decisions, including the Bcl-2 family of apoptosis-regulating proteins, is crucial to the EBV cycle of infection. Here, we show that BIK (also known as NBK), which encodes a proapoptotic "sensitizer" protein, is repressed by the EBNA2-driven Lat III program but not the Lat I program. BIK repression occurred soon after infection of primary B cells by EBV but not by a recombinant EBV in which the EBNA2 gene had been knocked out. Ectopic BIK induced apoptosis in Lat III cells by a mechanism dependent on its BH3 domain and the activation of caspases. We show that EBNA2 represses BIK in EBV-negative B-cell lymphoma-derived cell lines and that this host-virus interaction can inhibit the proapoptotic effect of transforming growth factor ß1 (TGF-ß1), a key physiological mediator of B-cell homeostasis. Reduced levels of TGF-ß1-associated regulatory SMAD proteins were bound to the BIK promoter in response to EBV Lat III or ectopic EBNA2. These data are evidence of an additional mechanism used by EBV to promote B-cell survival, namely, the transcriptional repression of the BH3-only sensitizer BIK. IMPORTANCE: Over 90% of adult humans are infected with the Epstein-Barr virus (EBV). EBV establishes a lifelong silent infection, with its DNA residing in small numbers of blood B cells that are a reservoir from which low-level virus reactivation and shedding in saliva intermittently occur. Importantly, EBV DNA is found in some B-cell-derived tumors in which viral genes play a key role in tumor cell emergence and progression. Here, we report for the first time that EBV can shut off a B-cell gene called BIK. When activated by a molecular signal called transforming growth factor ß1 (TGF-ß1), BIK plays an important role in killing unwanted B cells, including those infected by viruses. We describe the key EBV-B-cell molecular interactions that lead to BIK shutoff. These findings further our knowledge of how EBV prevents the death of its host cell during infection. They are also relevant to certain posttransplant lymphomas where unregulated cell growth is caused by EBV genes.

Expression of Angiogenic Factors in Patients With Sporadic Small Bowel Angiodysplasia.

GOALS: To identify putative angiogenic factors associated with sporadic small bowel angiodysplasia (SBA). BACKGROUND: SBAs account for 50% of obscure gastrointestinal bleeding and due to delays in diagnosis and ineffective treatments, are associated with high levels of morbidity and mortality. Treatment development is impeded by a limited knowledge of the pathophysiology behind SBA formation. STUDY: We identified patients with definite sporadic SBA, and fecal immunochemical-negative controls were recruited from our institution's colorectal cancer screening program. Serum levels of VEGF, endoglin, Angiopoietin-2 (Ang-2), PDGF, Angiopoietin-1 (Ang-1), and TNF-α were measured using commercially available enzyme-linked immunosorbent assay kits. On the basis of serum results, we measured gene expression of target angiogenic factors in small bowel biopsy samples from angiodysplasias and unaffected tissue by quantitative PCR assessment. RESULTS: Serum samples were analyzed from 40 SBA patients and 40 controls. Median serum levels of Ang-2 were significantly higher in patients than controls with levels of Ang-1 and TNF-α significantly lower. There were no differences in serum levels of VEGF, endoglin, or PDGF. Gene expression levels of Ang-1, Ang-2, and their receptor Tie2 were all significantly higher in biopsies from areas of angiodysplasia compared with normal small bowel. CONCLUSIONS: This study, the first to explore the role of angiogenic factors in SBA, has identified a positive association between SBA and the Angiopoietin pathway, with increased serum and mucosal expression of Ang-2, which could potentially be used as a serum biomarker and future therapeutic target to improve outcome in affected patients.

Vitamin D Status Is Associated with Intestinal Inflammation as Measured by Fecal Calprotectin in Crohn's Disease in Clinical Remission.

BACKGROUND: Vitamin D, as potential immune modulator, has been implicated as an environmental risk factor for Crohn's disease (CD). Vitamin D status may be associated with disease risk, severity, activity, and progression. While associations between circulating 25OHD and markers of disease activity and inflammation in CD have been reported, the results are inconsistent. AIM: To determine the association between vitamin D status and markers of disease activity and inflammation in CD. METHODS: One hundred and nineteen CD patients' active and inactive diseases were enrolled in the cross-sectional study. Subject demographics and clinical data were collected. A serum sample was collected for 25OHD and CRP analysis, and a stool sample was collected for fecal calprotectin (FC) measurement. RESULTS: The mean serum 25OHD concentration of the group was 59.8 (24.9) nmol/L. After controlling for confounding variables, serum 25OHD inversely correlated with FC (r = -0.207, P = 0.030), particularly among those in clinical remission (r = -0.242, P = 0.022). The association between FC and 25OHD was further confirmed by linear regression (r = 31.3 %, P < 0.001). FC was lower in patients with 25OHD levels ≥75 nmol/L compared with levels <25 nmol/L [FC: 32.2 (16.3-98.2) vs 100.0 (34.4-213.5) µg/g, P = 0.004]. In the current study, however, 25OHD was not significantly associated with either CRP or CDAI. CONCLUSION: Circulating 25OHD was significantly inversely associated with intestinal inflammation as determined by FC in CD. Subgroup analysis confirmed the association among those in clinical remission, but not in those with active disease. 25OHD was not associated with disease activity score (CDAI) or systemic inflammation (CRP). Vitamin D intervention studies are warranted to determine whether raising serum 25OHD levels in patients with CD may reduce intestinal inflammation as measured by FC.

The second Irish Helicobacter pylori Working Group consensus for the diagnosis and treatment of Helicobacter pylori infection in adult patients in Ireland.

BACKGROUND: There has been an increase in resistance to many of the antimicrobials used to treat Helicobacter pylori ( H. pylori ) nationally and internationally. Primary clarithromycin resistance and dual clarithromycin and metronidazole resistance are high in Ireland. These trends call for an evaluation of best-practice management strategies. OBJECTIVE: The objective of this study was to revise the recommendations for the management of H. pylori infection in adult patients in the Irish healthcare setting. METHODS: The Irish H. pylori working group (IHPWG) was established in 2016 and reconvened in 2023 to evaluate the most up-to-date literature on H. pylori diagnosis, eradication rates and antimicrobial resistance. The 'GRADE' approach was then used to rate the quality of available evidence and grade the resulting recommendations. RESULTS: The Irish H. pylori working group agreed on 14 consensus statements. Key recommendations include (1) routine antimicrobial susceptibility testing to guide therapy is no longer recommended other than for clarithromycin susceptibility testing for first-line treatment (statements 6 and 9), (2) clarithromycin triple therapy should only be prescribed as first-line therapy in cases where clarithromycin susceptibility has been confirmed (statement 9), (3) bismuth quadruple therapy (proton pump inhibitor, bismuth, metronidazole, tetracycline) is the recommended first-line therapy if clarithromycin resistance is unknown or confirmed (statement 10), (4) bismuth quadruple therapy with a proton pump inhibitor, levofloxacin and amoxicillin is the recommended second-line treatment (statement 11) and (5) rifabutin amoxicillin triple therapy is the recommend rescue therapy (statement 12). CONCLUSION: These recommendations are intended to provide the most relevant current best-practice guidelines for the management of H. pylori infection in adults in Ireland.

Helicobacter pylori: High dose amoxicillin does not improve primary or secondary eradication rates in an Irish cohort.

BACKGROUND: Helicobacter pylori (H. pylori) eradication rates have fallen globally, likely in large part due to increasing antibiotic resistance to traditional therapy. In areas of high clarithromycin and metronidazole resistance such as ours, Maastricht VI guidelines suggest high dose amoxicillin dual therapy (HDADT) can be considered, subject to evidence for local efficacy. In this study we assess efficacy of HDADT therapy for H. pylori eradication in an Irish cohort. AIM: To assess the efficacy of HDADT therapy for H. pylori eradication in an Irish cohort as both first line, and subsequent therapy for patients diagnosed with H. pylori. METHODS: All patients testing positive for H. pylori in a tertiary centre were treated prospectively with HDADT (amoxicillin 1 g tid and esomeprazole 40 mg bid × 14 d) over a period of 8 months. Eradication was confirmed with Urea Breath Test at least 4 wk after cessation of therapy. A delta-over-baseline > 4% was considered positive. Patient demographics and treatment outcomes were recorded, analysed and controlled for basic demographics and prior H. pylori treatment. RESULTS: One hundred and ninety-eight patients were identified with H. pylori infection, 10 patients were excluded due to penicillin allergy and 38 patients refused follow up testing. In all 139 were included in the analysis, 55% (n = 76) were female, mean age was 46.6 years. Overall, 93 (67%) of patients were treatment-naïve and 46 (33%) had received at least one previous course of treatment. The groups were statistically similar. Self-reported compliance with HDADT was 97%, mild side-effects occurred in 7%. There were no serious adverse drug reactions. Overall the eradication rate for our cohort was 56% (78/139). Eradication rates were worse for those with previous treatment [43% (20/46) vs 62% (58/93), P = 0.0458, odds ratio = 2.15]. Age and Gender had no effect on eradication status. CONCLUSION: Overall eradication rates with HDADT were disappointing. Despite being a simple and possibly better tolerated regime, these results do not support its routine use in a high dual resistance country. Further investigation of other regimens to achieve the > 90% eradication target is needed.

Role of compliance in Helicobacter pylori eradication treatment: Results of the European Registry on H. pylori management.

BACKGROUND: Adherence to Helicobacter pylori (H. pylori) eradication treatment is a cornerstone for achieving adequate treatment efficacy. OBJECTIVE: To determine which factors influence compliance with treatment. METHODS: A systematic prospective non-interventional registry (Hp-EuReg) of the clinical practice of European gastroenterologists. Compliance was considered adequate if ≥90% drug intake. Data were collected until September 2021 using the AEG-REDCap e-CRF and were subjected to quality control. Modified intention-to-treat analyses were performed. Multivariate analysis carried out the factors associated with the effectiveness of treatment and compliance. RESULTS: Compliance was inadequate in 646 (1.7%) of 38,698 patients. The non-compliance rate was higher in patients prescribed longer regimens (10-, 14-days) and rescue treatments, patients with uninvestigated dyspepsia/functional dyspepsia, and patients reporting adverse effects. Prevalence of non-adherence was lower for first-line treatment than for rescue treatment (1.5% vs. 2.2%; p < 0.001). Differences in non-adherence in the three most frequent first-line treatments were shown: 1.1% with proton pump inhibitor + clarithromycin + amoxicillin; 2.3% with proton pump inhibitor clarithromycin amoxicillin metronidazole; and 1.8% with bismuth quadruple therapy. These treatments were significantly more effective in compliant than in non-compliant patients: 86% versus 44%, 90% versus 71%, and 93% versus 64%, respectively (p < 0.001). In the multivariate analysis, the variable most significantly associated with higher effectiveness was adequate compliance (odds ratio, 6.3 [95%CI, 5.2-7.7]; p < 0.001). CONCLUSIONS: Compliance with Helicobacter pylori eradication treatment is very good. Factors associated with poor compliance include uninvestigated/functional dyspepsia, rescue-treatment, prolonged treatment regimens, the presence of adverse events, and the use of non-bismuth sequential and concomitant treatment. Adequate treatment compliance was the variable most closely associated with successful eradication.

A role for TLR4 in Clostridium difficile infection and the recognition of surface layer proteins.

Clostridium difficile is the etiological agent of antibiotic-associated diarrhoea (AAD) and pseudomembranous colitis in humans. The role of the surface layer proteins (SLPs) in this disease has not yet been fully explored. The aim of this study was to investigate a role for SLPs in the recognition of C. difficile and the subsequent activation of the immune system. Bone marrow derived dendritic cells (DCs) exposed to SLPs were assessed for production of inflammatory cytokines, expression of cell surface markers and their ability to generate T helper (Th) cell responses. DCs isolated from C3H/HeN and C3H/HeJ mice were used in order to examine whether SLPs are recognised by TLR4. The role of TLR4 in infection was examined in TLR4-deficient mice. SLPs induced maturation of DCs characterised by production of IL-12, TNFα and IL-10 and expression of MHC class II, CD40, CD80 and CD86. Furthermore, SLP-activated DCs generated Th cells producing IFNγ and IL-17. SLPs were unable to activate DCs isolated from TLR4-mutant C3H/HeJ mice and failed to induce a subsequent Th cell response. TLR4⁻/⁻ and Myd88⁻/⁻, but not TRIF⁻/⁻ mice were more susceptible than wild-type mice to C. difficile infection. Furthermore, SLPs activated NFκB, but not IRF3, downstream of TLR4. Our results indicate that SLPs isolated from C. difficile can activate innate and adaptive immunity and that these effects are mediated by TLR4, with TLR4 having a functional role in experimental C. difficile infection. This suggests an important role for SLPs in the recognition of C. difficile by the immune system.

Tribbles 3: a novel regulator of TLR2-mediated signaling in response to Helicobacter pylori lipopolysaccharide.

Helicobacter pylori causes chronic gastritis, peptic ulcers, and gastric carcinoma. Gastric epithelial cells provide the first point of contact between H. pylori and the host. TLRs present on these cells recognize various microbial products, resulting in the initiation of innate immunity. Although previous reports investigated TLR signaling in response to intact H. pylori, the specific contribution of H. pylori LPS with regard to functional genomics and cell-signaling events has not been defined. This study set out to define downstream signaling components and altered gene expression triggered by H. pylori LPS and to investigate the role of the signaling protein tribbles 3 (TRIB3) during the TLR-mediated response to H. pylori LPS. Cotransfections using small interfering RNA and dominant-negative constructs demonstrated that H. pylori LPS functions as a classic TLR2 ligand by signaling through pathways involving the key TLR signaling components MyD88 adaptor-like, MyD88, IRAK1, IRAK4, TNFR-associated factor 6, IκB kinase ß, and IκBα. Microarray analysis, real-time PCR, and ELISA revealed the induction of a discrete pattern of chemokines as a direct effect of LPS:TLR2 signaling. H. pylori infection was associated with decreased expression of TRIB3 in human gastric epithelial cell lines and tissue samples. Additionally, H. pylori decreased expression of C/EBP homologous protein and activating transcription factor 4, the transcription factors involved in the induction of TRIB3 expression. Furthermore, knockdown of TRIB3 and C/EBP homologous protein enhanced TLR2-mediated NF-κB activation and chemokine induction in response to H. pylori LPS. Thus, modulation of TRIB3 by H. pylori and/or its products may be an important mechanism during H. pylori-associated pathogenesis.

Strategies for the Purification of Membrane Proteins.

Membrane proteins account for approximately 30% of the coding regions of all sequenced genomes, and they play crucial roles in many fundamental cell processes. However, there are relatively few membrane proteins with known three-dimensional structures. This is likely due to technical challenges associated with membrane protein extraction, solubilization, and purification. Membrane proteins are classified based on the level of interaction with membrane lipid bilayers, with peripheral membrane proteins associating non-covalently with the membrane, and integral membrane proteins associating more strongly by means of hydrophobic interactions. Generally speaking, peripheral membrane proteins can be purified by milder techniques than integral membrane proteins, with the latter's extraction requiring phospholipid bilayer disruption using detergents or organic solvents. In this chapter, important considerations for membrane protein purification are addressed, with a focus on the initial stages of membrane protein solubilization, where problems are most frequently encountered. Protocols are outlined for the extraction of peripheral membrane proteins, solubilization of integral membrane proteins, and sample clean-up and concentration.

Impact of test, vaccinate or remove protocol on home ranges and nightly movements of badgers a medium density population.

In the British Isles, the European badger (Meles meles) is thought to be the primary wildlife reservoir of bovine tuberculosis (bTB), an endemic disease in cattle. Test, vaccinate or remove ('TVR') of bTB test-positive badgers, has been suggested to be a potentially useful protocol to reduce bTB incidence in cattle. However, the practice of removing or culling badgers is controversial both for ethical reasons and because there is no consistent observed effect on bTB levels in cattle. While removing badgers reduces population density, it may also result in disruption of their social behaviour, increase their ranging, and lead to greater intra- and inter-species bTB transmission. This effect has been recorded in high badger density areas, such as in southwest England. However, little is known about how TVR affects the behaviour and movement of badgers within a medium density population, such as those that occur in Northern Ireland (NI), which the current study aimed to examine. During 2014-2017, badger ranging behaviours were examined prior to and during a TVR protocol in NI. Nightly distances travelled by 38 individuals were determined using Global Positioning System (GPS) measurements of animal tracks and GPS-enhanced dead-reckoned tracks. The latter was calculated using GPS, tri-axial accelerometer and tri-axial magnetometer data loggers attached to animals. Home range and core home range size were measured using 95% and 50% autocorrelated kernel density estimates, respectively, based on location fixes. TVR was not associated with measured increases in either distances travelled per night (mean = 3.31 ± 2.64 km) or home range size (95% mean = 1.56 ± 0.62 km2, 50% mean = 0.39 ± 0.62 km2) over the four years of study. However, following trapping, mean distances travelled per night increased by up to 44% eight days post capture. Findings differ from those observed in higher density badger populations in England, in which badger ranging increased following culling. Whilst we did not assess behaviours of individual badgers, possible reasons why no differences in home range size were observed include higher inherent 'social fluidity' in Irish populations whereby movements are less restricted by habitat saturation and/or that the numbers removed did not reach a threshold that might induce increases in ranging behaviour. Nevertheless, short-term behavioural disruption from trapping was observed, which led to significant increases in the movements of individual animals within their home range. Whether or not TVR may alter badger behaviours remains to be seen, but it would be better to utilise solutions such as oral vaccination of badgers and/or cattle as well as increased biosecurity to limit bTB transmission, which may be less likely to cause interference and thereby reduce the likelihood of bTB transmission.