Peritoneal Immunity in Liver Disease

The peritoneum represents a confined microenvironment that has an emerging role as a distinct immunological compartment. In health, this niche is mainly populated by a heterogenous group of macrophages and T lymphocytes but also Natural Killer cells and B lymphocytes. Together they are crucial for immunological surveillance, clearance of infection and resolution of inflammation. Development of ascites is a defining feature of decompensated liver cirrhosis, and spontaneous bacterial peritonitis is the most frequent bacterial infection occurring in this patient group. Recent studies of ascitic fluid have revealed quantitative, phenotypic and functional differences in both innate and adaptive immune cells compared to the healthy state. This review summarises current knowledge of these alterations and explores how the peritoneum in chronic liver disease is simultaneously an immunologically compromised site and yet capable of provoking an intense inflammatory response. A better understanding of this might enable identification of new therapeutic targets aimed to rebalance the peritoneal immunity and reduce the reliance on antimicrobials in an era of increasing antimicrobial resistance

Drop-out rate from the liver transplant waiting list due to HCC progression in HCV-infected patients treated with direct acting antivirals.

BACKGROUND & AIM: concerns about an increased hepatocellular carcinoma (HCC) recurrence rate following directly acting antiviral (DAA) therapy in cirrhotic patients with a prior complete oncological response have been raised. Data regarding the impact of HCV-treatment with DAAs on waiting list drop-out rates in patients with active HCC and HCV-related cirrhosis awaiting liver transplantation (LT) are lacking. MATERIALS AND METHODS: HCV-HCC patients listed for LT between January 2015 and May 2016 at Padua Liver Transplant Centre were considered eligible for the study. After enrollment patients were divided into 2 groups, depending on whether they underwent DAAs treatment while awaiting LT or not. For each patient clinical, serological and virological data were collected. HCC characteristics were radiologically evaluated at baseline and during follow-up (FU). For transplanted patients, pathological assessment of the explants was performed and recurrence-rates were calculated. RESULTS: twenty-three patients treated with DAAs and 23 controls were enrolled. HCC characteristics at time of LT-listing were comparable between the 2 groups. Median FU was 10 and 7 months, respectively, during which 2/23 (8.7%) and 1/23 (4.3%) drop-out events due to HCC-progression were registered (p = 0.9). No significant differences in terms of radiological progression were highlighted (p = 0.16). Nine out of 23 cases (39%) and 14/23 (61%) controls underwent LT, and histopathological analysis showed no differences in terms of median number and total tumor volume of HCC nodules, tumor differentiation or microvascular invasion. During post-LT FU, 1/8 DAAs treated patient (12,5%) and 1/12 control (8,3%) experienced HCC recurrence (p = 0.6). CONCLUSIONS: Viral eradication does not seem to be associated with an increased risk of drop-out due to neoplastic progression in HCV-HCC patients awaiting LT

TLR9 polymorphisms in African populations: no association with severe malaria, but evidence of cis-variants acting on gene expression

BACKGROUND: During malaria infection the Toll-like receptor 9 (TLR9) is activated through induction with plasmodium DNA or another malaria motif not yet identified. Although TLR9 activation by malaria parasites is well reported, the implication to the susceptibility to severe malaria is not clear. The aim of this study was to assess the contribution of genetic variation at TLR9 to severe malaria. METHODS: This study explores the contribution of TLR9 genetic variants to severe malaria using two approaches. First, an association study of four common single nucleotide polymorphisms was performed on both family- and population-based studies from Malawian and Gambian populations (n>6000 individual). Subsequently, it was assessed whether TLR9 expression is affected by cis-acting variants and if these variants could be mapped. For this work, an allele specific expression (ASE) assay on a panel of HapMap cell lines was carried out. RESULTS: No convincing association was found with polymorphisms in TLR9 for malaria severity, in either Gambian or Malawian populations, using both case-control and family based study designs. Using an allele specific expression assay it was observed that TLR9 expression is affected by cis-acting variants, these results were replicated in a second experiment using biological replicates. CONCLUSION: By using the largest cohorts analysed to date, as well as a standardized phenotype definition and study design, no association of TLR9 genetic variants with severe malaria was found. This analysis considered all common variants in the region, but it is remains possible that there are rare variants with association signals. This report also shows that TLR9 expression is potentially modulated through cis-regulatory variants, which may lead to differential inflammatory responses to infection between individuals

Cystic fibrosis newborn screening: the importance of bloodspot sample quality

Objective Wales has an immunoreactive trypsin (IRT)-DNA cystic fibrosis (CF) newborn screening (NBS) programme. Most CF NBS false negative cases are due to an IRT concentration below the screening threshold. The accuracy of IRT results is dependent on the quality of the dried bloodspot (DBS) sample. The aim of this study was to determine the cause of false negative cases in CF NBS and their relationship to DBS quality. Design Longitudinal birth cohort. Setting Wales 1996–2016. Patients Children with CF. Interventions Identification of all CF patients with triangulation of multiple data sources to detect false negative cases. Main outcome measures False negative cases. Results Over 20 years, 673 952 infants were screened and 239 were diagnosed with CF (incidence 1:2819). The sensitivity of the programme was 0.958, and positive predictive value was 0.476. Eighteen potential false negatives were identified, of whom eight were excluded: four screened outside Wales, two had complex comorbidities, no identified cystic fibrosis transmembrane conductance regulator (CFTR) variants on extended analysis and thus not considered to have CF and two were diagnosed after their 16th birthday. Of the 10 false negatives, 9 had a low DBS IRT and at least one common CFTR variant and thus should have received a sweat test under the programme. DBS cards were available for inspection for five of the nine false negative cases—all were classified as small/insufficient or poor quality. Conclusions The majority of false negatives had a low bloodspot IRT, and this was associated with poor quality DBS. The optimal means to improve the sensitivity of our CF NBS programme would be to improve DBS sample quality

Phenotypic and genotypic adaptations in Pseudomonas aeruginosa biofilms following long-term exposure to an alginate oligomer therapy

Chronic Pseudomonas aeruginosa lung infections in cystic fibrosis (CF) evolve to generate environmentally adapted biofilm communities, leading to increased patient morbidity and mortality. OligoG CF-5/20, a low-molecular-weight inhaled alginate oligomer therapy, is currently in phase IIb/III clinical trials in CF patients. Experimental evolution of P. aeruginosa in response to OligoG CF-5/20 was assessed using a bead biofilm model allowing continuous passage (45 days; ∼245 generations). Mutants isolated after OligoG CF-5/20 treatment typically had a reduced biofilm-forming ability and altered motility profile. Genotypically, OligoG CF-5/20 provided no selective pressure on genomic mutations within morphotypes. Chronic exposure to azithromycin, a commonly prescribed antibiotic in CF patients, with or without OligoG CF-5/20 in the biofilm evolution model also had no effect on rates of resistance acquisition. Interestingly, however, cross-resistance to other antibiotics (e.g., aztreonam) was reduced in the presence of OligoG CF-5/20. Collectively, these findings show no apparent adverse effects from long-term exposure to OligoG CF-5/20, instead resulting in both fewer colonies with multidrug resistance (MDR)-associated phenotypes and improved antibiotic susceptibility of P. aeruginosa

Strategic treatment optimization for HCV (STOPHCV1): a randomised controlled trial of ultrashort duration therapy for chronic hepatitis C [version 1; peer review: awaiting peer review]

Background: The world health organization (WHO) has identified the need for a better understanding of which patients with hepatitis C virus (HCV) can be cured with ultrashort course HCV therapy. Methods: A total of 202 individuals with chronic HCV were randomised to fixed-duration shortened therapy (8 weeks) vs variable duration ultrashort strategies (VUS1/2). Participants not cured following first-line treatment were retreated with 12 weeks’ sofosbuvir/ledipasvir/ribavirin. The primary outcome was sustained virological response 12 weeks (SVR12) after first-line treatment and retreatment. Participants were factorially randomised to receive ribavirin with first-line treatment. Results: All evaluable participants achieved SVR12 overall (197/197, 100% [95% CI 98-100]) demonstrating non-inferiority between fixedduration and variable-duration strategies (difference 0% [95% CI - 3.8%, +3.7%], 4% pre-specified non-inferiority margin). First-line SVR12 was 91% [86%-97%] (92/101) for fixed-duration vs 48% [39%-57%] (47/98) for variable-duration, but was significantly higher for VUS2 (72% [56%-87%] (23/32)) than VUS1 (36% [25%-48%] (24/66)). Overall, first-line SVR12 was 72% [65%-78%] (70/101) without ribavirin and 68% [61%-76%] (69/98) with ribavirin (p=0.48). At treatment failure, the emergence of viral resistance was lower with ribavirin (12% [2%-30%] (3/26)) than without (38% [21%-58%] (11/29), p=0.01). Conclusions: Unsuccessful first-line short-course therapy did not compromise retreatment with sofosbuvir/ledipasvir/ribavirin (100% SVR12). SVR12 rates were significantly increased when ultrashort treatment varied between 4-7 weeks rather than 4-6 weeks. Ribavirin significantly reduced resistance emergence in those failing first-line therapy. ISRCTN Registration: 37915093 (11/04/2016)

Telomere length in cystic fibrosis patients – Are patients with CF ageing too quickly?

Life expectancy for patients living with Cystic Fibrosis (CF) is increasing year on year and there is growing interest in the ageing process in CF. Telomeres are repetitive sequences of DNA that cap the ends of eukaryotic chromosomes and shorten with ongoing cell division, thus providing a marker of replicative history and biological ageing. We aimed to investigate whether telomere length as a function of age differs between patients with CF and healthy individuals and whether telomere length is associated with severity of the patient’s CF condition.Peripheral blood samples and demographic data were collected from 47 consenting patients (age 1 to 57 years) with CF attending their routine annual review appointment at the All Wales Adult CF Centre and Noah’s Ark Children’s’ Hospital in Cardiff, UK. Telomere length profiles were assessed from peripheral blood samples, using the high resolution single telomere length analysis technique (STELA) and compared to healthy control telomere length data.Patients with CF had significantly shorter telomere lengths than healthy individuals, when adjusting for age (

Mucin structural interactions with an alginate oligomer mucolytic in cystic fibrosis sputum

Cystic fibrosis (CF) is an autosomal recessive, life-limiting condition characterized by progressive lung disease, which is a major cause of morbidity and mortality for these patients. The inhalation therapy, OligoG CF-5/20, is a low molecular weight (mean Mn 3200 g/mol) alginate oligomer, with a high guluronic acid content (>85%). The ability of OligoG CF-5/20 to enhance the activity of antimicrobial/antibiotic therapies, modify the rheological properties of CF sputum and interact with mucin, has previously been shown. To further characterize the physicochemical interactions of OligoG CF-5/20 with CF sputum, Fourier-transform infrared (FTIR) spectroscopy was used to analyze ex vivo sputum samples from adolescent CF patients (n = 13) following treatment with 0.2% OligoG CF-5/20. FTIR analysis confirmed the interaction of OligoG CF-5/20 with mucin glycans in CF sputum, which showed a shift in wavenumber from 1078 cm−1 to 1070 cm-1 and subsequent loss of the 1053 cm−1 peak in the OligoG CF-5/20 treated samples. OligoG CF-5/20 interaction with key terminal moieties in mucin were also evident, with a significant change in sulphation at wavenumber 1116 cm−1, suggesting a link with sulphated Lewis x antigen. There were also significant shifts at wavenumber 1637 cm-1 indicative of β-sheet conformational changes in the mucin peptide caused by action of OligoG. The alterations in charge of glycan and mucin structures support previous observations wherein OligoG CF-5/20 modifies the viscoelastic properties of CF sputum. These findings suggest a possible mechanism of action for the rheological changes observed with this novel therapy