Heart failure in sub-Saharan Africa: review of the aetiology of heart failure and the role of point-of-care biomarker diagnostics

Within Africa, the burden of heart failure is significant. This arises from the increase in cardiovascular disease and associated risk factors such as hypertension and diabetes, as well as causes of heart failure which are particular to sub-Saharan Africa, such as endomyocardial fibrosis. The lack of access to echocardiography and other imaging modalities, from a cost and technical perspective, combined with the predominantly rural nature of many countries with poor transport links, means that the vast majority of people never obtain an appropriate diagnosis. Similarly, research has been limited on the causes and treatment of heart failure in Africa and in particular endemic causes such as EMF and rheumatic heart disease. This review outlines the burden of heart failure in Africa and highlights the opportunity to expand diagnosis through the use of biomarkers, in particular natriuretic peptides. This builds on the success of point-of-care testing in human immunodeficiency virus and tuberculosis which have been extensively deployed in community settings in Africa.

Efficacy and safety of lumacaftor/ivacaftor combination therapy in patients with cystic fibrosis homozygous for Phe508del CFTR by pulmonary function subgroup

Background Lumacaftor/ivacaftor combination therapy demonstrated clinical benefits inpatients with cystic fibrosis homozygous for the Phe508del CFTR mutation.Pretreatment lung function is a confounding factor that potentially impacts the efficacyand safety of lumacaftor/ivacaftor therapy. Methods Two multinational, randomised, double-blind, placebo-controlled, parallelgroupPhase 3 studies randomised patients to receive placebo or lumacaftor (600 mgonce daily [qd] or 400 mg every 12 hours [q12h]) in combination with ivacaftor (250 mgq12h) for 24 weeks. Prespecified analyses of pooled efficacy and safety data by lungfunction, as measured by percent predicted forced expiratory volume in 1 second(ppFEV1), were performed for patients with baseline ppFEV1 <40 (n=81) and ≥40(n=1016) and screening ppFEV1 <70 (n=730) and ≥70 (n=342). These studies wereregistered with ClinicalTrials.gov (NCT01807923 and NCT01807949). Findings The studies were conducted from April 2013 through April 2014.Improvements in the primary endpoint, absolute change from baseline at week 24 inppFEV1, were observed with both lumacaftor/ivacaftor doses in the subgroup withbaseline ppFEV1 <40 (least-squares mean difference versus placebo was 3∙7 and 3.3percentage points for lumacaftor 600 mg qd/ivacaftor 250 mg q12h and lumacaftor 400mg q12h/ivacaftor 250 mg q12h, respectively [p<0∙05] and in the subgroup with baselineppFEV1 ≥40 (3∙3 and 2∙8 percentage points, respectively [p<0∙001]). Similar absoluteimprovements versus placebo in ppFEV1 were observed in subgroups with screening 4ppFEV1 <70 (3∙3 and 3∙3 percentage points for lumacaftor 600 mg qd/ivacaftor 250 mgq12h and lumacaftor 400 mg q12h/ivacaftor 250 mg q12h, respectively [p<0∙001]) and≥70 (3∙3 and 1∙9 percentage points, respectively [p=0.002] and [p=0∙079]). Increases inBMI and reduction in number of pulmonary exacerbation events were observed in bothLUM/IVA dose groups vs placebo across all lung function subgroups. Treatment wasgenerally well tolerated, although the incidence of some respiratory adverse events washigher with active treatment than with placebo. Interpretation Lumacaftor/ivacaftor combination therapy benefits patients homozygousfor Phe508del CFTR who have varying degrees of lung function impairment. Funding Vertex Pharmaceuticals Incorporated.

Epigenetics and the overhealing wound: the role of DNA methylation in fibrosis

Fibrosis is a progressive and potentially fatal process that can occur in numerous organ systems. Characterised by the excessive deposition of extracellular matrix proteins such as collagens and fibronectin, fibrosis affects normal tissue architecture and impedes organ function. Although a considerable amount of research has focused on the mechanisms underlying disease pathogenesis, current therapeutic options do not directly target the pro-fibrotic process. As a result, there is a clear unmet clinical need to develop new agents. Novel findings implicate a role for epigenetic modifications contributing to the progression of fibrosis by alteration of gene expression profiles. This review will focus on DNA methylation; its association with fibroblast differentiation and activation and the consequent buildup of fibrotic scar tissue. The potential use of therapies that modulate this epigenetic pathway for the treatment of fibrosis in several organ systems is also discussed.

Epigenetic Therapy for the Treatment of Hypertension-Induced Cardiac Hypertrophy and Fibrosis

BACKGROUND: The development of heart failure is associated with changes in the size, shape, and structure of the heart that has a negative impact on cardiac function. These pathological changes involve excessive extracellular matrix deposition within the myocardial interstitium and myocyte hypertrophy. Alterations in fibroblast phenotype and myocyte activity are associated with reprogramming of gene transcriptional profiles that likely requires epigenetic alterations in chromatin structure. The aim of our work was to investigate the potential of a currently licensed anticancer epigenetic modifier as a treatment option for cardiac diseases associated with hypertension-induced cardiac hypertrophy and fibrosis.

METHODS AND RESULTS: The effects of DNA methylation inhibition with 5-azacytidine (5-aza) were examined in a human primary fibroblast cell line and in a spontaneously hypertensive rat (SHR) model. The results from this work allude to novel in vivo antifibrotic and antihypertrophic actions of 5-aza. Administration of the DNA methylation inhibitor significantly improved several echocardiographic parameters associated with hypertrophy and diastolic dysfunction. Myocardial collagen levels and myocyte size were reduced in 5-aza-treated SHRs. These findings are supported by beneficial in vitro effects in cardiac fibroblasts. Collagen I, collagen III, and α-smooth muscle actin were reduced in a human ventricular cardiac fibroblast cell line treated with 5-aza.

CONCLUSION: These findings suggest a role for epigenetic modifications in contributing to the profibrotic and hypertrophic changes evident during disease progression. Therapeutic intervention with 5-aza demonstrated favorable effects highlighting the potential use of this epigenetic modifier as a treatment option for cardiac pathologies associated with hypertrophy and fibrosis.

Hypoxia-induced epigenetic modifications are associated with cardiac tissue fibrosis and the development of a myofibroblast-like phenotype

Ischemia caused by coronary artery disease and myocardial infarction leads to aberrant ventricular remodeling and cardiac fibrosis. This occurs partly through accumulation of gene expression changes in resident fibroblasts, resulting in an overactive fibrotic phenotype. Long-term adaptation to a hypoxic insult is likely to require significant modification of chromatin structure in order to maintain the fibrotic phenotype. Epigenetic changes may play an important role in modulating hypoxia-induced fibrosis within the heart. Therefore, the aim of the study was to investigate the potential pro-fibrotic impact of hypoxia on cardiac fibroblasts and determine whether alterations in DNA methylation could play a role in this process. This study found that within human cardiac tissue, the degree of hypoxia was associated with increased expression of collagen 1 and alpha-smooth muscle actin (ASMA). In addition, human cardiac fibroblast cells exposed to prolonged 1% hypoxia resulted in a pro-fibrotic state. These hypoxia-induced pro-fibrotic changes were associated with global DNA hypermethylation and increased expression of the DNA methyltransferase (DNMT) enzymes DNMT1 and DNMT3B. Expression of these methylating enzymes was shown to be regulated by hypoxia-inducible factor (HIF)-1α. Using siRNA to block DNMT3B expression significantly reduced collagen 1 and ASMA expression. In addition, application of the DNMT inhibitor 5-aza-2'-deoxycytidine suppressed the pro-fibrotic effects of TGFβ. Epigenetic modifications and changes in the epigenetic machinery identified in cardiac fibroblasts during prolonged hypoxia may contribute to the pro-fibrotic nature of the ischemic milieu. Targeting up-regulated expression of DNMTs in ischemic heart disease may prove to be a valuable therapeutic approach.

Mechanical stretch up-regulates the B-type natriuretic peptide system in human cardiac fibroblasts:a possible defense against transforming growth factor-β mediated fibrosis

BACKGROUND: Mechanical overload of the heart is associated with excessive deposition of extracellular matrix proteins and the development of cardiac fibrosis. This can result in reduced ventricular compliance, diastolic dysfunction, and heart failure. Extracellular matrix synthesis is regulated primarily by cardiac fibroblasts, more specifically, the active myofibroblast. The influence of mechanical stretch on human cardiac fibroblasts' response to pro-fibrotic stimuli, such as transforming growth factor beta (TGFβ), is unknown as is the impact of stretch on B-type natriuretic peptide (BNP) and natriuretic peptide receptor A (NPRA) expression. BNP, acting via NPRA, has been shown to play a role in modulation of cardiac fibrosis.

METHODS AND RESULTS: The effect of cyclical mechanical stretch on TGFβ induction of myofibroblast differentiation in primary human cardiac fibroblasts and whether differences in response to stretch were associated with changes in the natriuretic peptide system were investigated. Cyclical mechanical stretch attenuated the effectiveness of TGFβ in inducing myofibroblast differentiation. This finding was associated with a novel observation that mechanical stretch can increase BNP and NPRA expression in human cardiac fibroblasts, which could have important implications in modulating myocardial fibrosis. Exogenous BNP treatment further reduced the potency of TGFβ on mechanically stretched fibroblasts.

CONCLUSION: We postulate that stretch induced up-regulation of the natriuretic peptide system may contribute to the observed reduction in myofibroblast differentiation.

Sarcoidosis, alveolar β-actin and pulmonary fibrosis

BACKGROUND: Sarcoidosis is a multisystem granulomatous disease of unknown aetiology. Proteins present within the alveolar space early in sarcoidosis disease may provide an insight into novel mechanisms for the development of fibrotic disease and in particular pulmonary fibrosis.

METHODS: A modified two-dimensional difference gel electrophoresis protocol was applied to the human bronchoalveolar lavage fluid (hBALF) of four patients with non-persistent pulmonary interstitial disease at 4-year follow-up (defined as mild disease) and four patients who developed pulmonary interstitial disease at 4-year follow-up (defined as severe disease). The protein β-actin was identified by LC-MS/MS from a preparative gel and found to be significantly elevated in early lavages from the severe disease group. To look at the potential pro-fibrotic effects of this protein, primary human pulmonary fibroblasts (CCD-19Lu) were treated with recombinant β-actin following which qPCR and ELISA assays were used to measure any effects.

RESULTS: We found that β-actin levels were significantly elevated in early hBALF samples in patients who subsequently developed severe disease when compared to the mild group. Treating primary human pulmonary fibroblasts with recombinant β-actin led to enhanced gene expression of the pro-fibrotic markers alpha smooth muscle actin and collagen 1 as well as the increased secretion of interleukin-13 and metalloproteinases 3 and 9.

CONCLUSION: Free β-actin within the lungs of sarcoidosis patients potentially may contribute to disease pathogenesis particularly in the context of abnormal remodelling and the development of pulmonary fibrosis.

Connectivity Mapping (ssCMap) To Predict A20 Inducing Drugs: Anti-inflammatory Action In Cystic Fibrosis

Cystic Fibrosis (CF) lung disease is characterised by a chronic and exaggerated inflammation in the airways. Despite recent developments to therapeutically overcome the underlying functional defect in CFTR (cystic fibrosis transmembrane conductance regulator), there is still an unmet need to also normalise the inflammatory response. The prolonged and heightened inflammatory response in CF is in part mediated by a lack of intrinsic downregulation of the pro-inflammatory NF-kB pathway. We have previously identified reduced expression of the NF-kB down-regulator A20 in CF as a key target to normalise the inflammatory response. Here we have used publically available gene array expression data together with sscMap (statistically significant connections’map)to successfully predict drugs already licensed for the use in humans to induce A20 mRNA and protein expression and thereby reduce inflammation. The effect of the predicted drugs on A20 and NFkB (p65) expression (mRNA) as well as pro-inflammatory cytokine release (IL-8) in the presence and absence of bacterial LPS was shown in bronchial epithelial cells lines (16HBE14o-, CFBE41o-) and in primary nasal epithelial cells (PNECs) from patients with CF (Phe508del homozygous) and non-CF controls. Additionally, the specificity of the drug action on A20 was confirmed using cell lines with TNFAIP3 (A20) knockdown (siRNA). We also show that the A20 inducing effect of ikarugamycin and quercetin is lower in CF derived airway epithelial cells than in non-CF cells.

Lack of Association Between Haptoglobin Phenotype and Cystic Fibrosis Outcomes

Haptoglobin (Hp), a heme-Iron chelator, has different isoforms which are associated with variable tendency toward infections: Hp 1-1, Hp 2-1, and Hp 2-2. Cystic fibrosis (CF) outcomes are variable and influenced by genetic and environmental factors. The aim of this study was to determine whether Hp phenotype influenced disease severity in CF. One hundred forty-two CF patients from two centers were analyzed for Haptoglobin phenotype using gel electrophoresis of hemoglobin enriched serum. Clinical and microbiological data including bacterial colonization status, lung function, presence of CF-related diabetes and liver disease, rate of exacerbation, and mortality were compared between Hp phenotype groups. We found a trend toward less mucoid PA among Hp 2-2 (20.4 %) compared with Hp 1-1 and Hp 2-1 individuals (33.3 %), p = 0.317. Hp 2-2 individuals also had less antibiotic courses, and lower inflammatory markers without statistical significance. Haptoglobin phenotype is unlikely to be an important modifier of CF phenotype.

Lung clearance index in adults and children with cystic fibrosis

Background: Lung clearance index (LCI) has good clinimetric properties and an acceptable feasibility profile as a surrogate endpoint in Cystic Fibrosis (CF). Although most studies to date have been in children, increasing numbers of adults with CF also have normal spirometry. Further study of LCI as an endpoint in CF adults is required. Therefore, the purpose of this study was to determine the clinimetric properties of LCI over the complete age range of people with CF. Methods: Clinically stable adults and children with CF and age matched healthy controls were recruited. Results: LCI and spirometry data for 110 CF subjects and 61 controls were collected at a stable visit. CF Questionnaire-Revised (CFQ-R) was completed by 80/110 CF subjects. Fifty-six CF subjects completed a second stable visit. The LCI CV% was 4.1% in adults and 6.3% in children with CF. The coefficient of repeatability of LCI was 1.2 in adults and 1.3 in children. In both adults and children, LCI (AUCROC=0.93 and 0.84) had greater combined sensitivity and specificity to discriminate between people with CF and controls compared to FEV1 (AUCROC=0.88 and 0.60) and FEF25-75 (AUCROC=0.87 and 0.68). LCI correlated significantly with the CFQ-R treatment burden in adults (r=-0.37; p<0.01) and children (r=-0.50; p<0.01). Washout tests were successful in 90% of CF subjects and were perceived as comfortable and easy to perform in both adults and children. Conclusions: These data support the use of LCI as a surrogate outcome measure in CF clinical trials in adults as well as children.

Antimicrobial susceptibility of Pseudomonas aeruginosa isolated from cystic fibrosis patients through Northern Europe

Pseudomonas aeruginosa is a major cause of morbidity and mortality in cystic fibrosis patients. This study compares the antimicrobial susceptibility of 153 P. aeruginosa isolates from the United Kingdom (UK) (n=58), Belgium (n=44), and Germany (n=51) collected from 120 patients during routine visits over the 2006-2012 period. MICs were measured by broth microdilution. Genes encoding extended spectrum β-lactamases (ESBL), metallo-β-lactamases and carbapenemases were detected by PCR. Pulsed Field Gel Electrophoresis and Multi-Locus Sequence Typing were performed on isolates resistant to ≥ 3 antibiotic classes among penicillins/cephalosporins, carbapenems, fluoroquinolones, aminoglycosides, polymyxins. Based on EUCAST/CLSI breakpoints, susceptibility was ≤ 30%/≤ 40% (penicillins, ceftazidime, amikacin, ciprofloxacin), 44-48%/48-63% (carbapenems), 72%/72% (tobramycin), and 92%/78% (colistin) independently of patient's age. Sixty percent of strains were multidrug resistant (MDR; European Centre for Disease prevention and Control criteria). Genes encoding ESBL (most prevalent BEL, PER, GES, VEB, CTX-M, TEM, SHV, and OXA), metallo β-lactamases (VIM, IMP, NDM), or carbapenemases (OXA-48, KPC) were not detected. The Liverpool Epidemic Strain (LES) was prevalent in UK isolates only (75% of MDR isolates). Four MDR ST958 isolates were found spread over the three countries. The other MDR clones were evidenced in ≤ 3 isolates and localized in a single country. A new sequence type (ST2254) was discovered in one MDR isolate in Germany. Clonal and non-clonal isolates with different susceptibility profiles were found in 21 patients. Thus, resistance and MDR are highly prevalent in routine isolates from 3 countries, with carbapenem (meropenem), tobramycin and colistin remaining the most active drugs.

The role of anaerobic bacteria in the cystic fibrosis airway

PURPOSE OF REVIEW: Anaerobic bacteria are not only normal commensals, but are also considered opportunistic pathogens and have been identified as persistent members of the lower airway community in people with cystic fibrosis of all ages and stages of disease. Currently, the role of anaerobic bacteria in cystic fibrosis lower airway disease is not well understood. Therefore, this review describes the recent studies relating to the potential pathophysiological role(s) of anaerobes within the cystic fibrosis lungs.

RECENT FINDINGS: The most frequently identified anaerobic bacteria in the lower airways are common to both cystic fibrosis and healthy lungs. Studies have shown that in cystic fibrosis, the relative abundance of anaerobes fluctuates in the lower airways with reduced lung function and increased inflammation associated with a decreased anaerobic load. However, anaerobes found within the lower airways also produce virulence factors, may cause a host inflammatory response and interact synergistically with recognized pathogens.

SUMMARY: Anaerobic bacteria are potentially members of the airway microbiota in health but could also contribute to the pathogenesis of lower airway disease in cystic fibrosis via both direct and indirect mechanisms. A personalized treatment strategy that maintains a normal microbial community may be possible in the future.

Future trends in cystic fibrosis demography in 34 European countries

Median survival has increased in people with cystic fibrosis (CF) during the past six decades, which has led to an increased number of adults with CF. The future impact of changes in CF demographics has not been evaluated. The aim of this study was to estimate the number of children and adults with CF in 34 European countries by 2025. Data were obtained from the European Cystic Fibrosis Society Patient Registry. Population forecasts were performed for countries that have extensive CF population coverage and at least 4 years of longitudinal data by modelling future entering and exiting flows in registry cohorts. For the other countries, population projections were performed based on assumptions from knowledge of current CF epidemiology. Western European countries' forecasts indicate that an increase in the overall number of CF patients by 2025, by approximately 50%, corresponds to an increase by 20% and by 75% in children and adults, respectively. In Eastern European countries the projections suggest a predominant increase in the CF child population, although the CF adult population would also increase.It was concluded that a large increase in the adult CF population is expected in the next decade. A significant increase in adult CF services throughout Europe is urgently required.

Modular approach to select bacteriophages targeting Pseudomonas aeruginosa for their application to children suffering with cystic fibrosis

This review discusses the potential application of bacterial viruses (phage therapy) towards the eradication of antibiotic resistant Pseudomonas aeruginosa in children with cystic fibrosis (CF). In this regard, several potential relationships between bacteria and their bacteriophages are considered. The most important aspect that must be addressed with respect to phage therapy of bacterial infections in the lungs of CF patients is in ensuring the continuity of treatment in light of the continual occurrence of resistant bacteria. This depends on the ability to rapidly select phages exhibiting an enhanced spectrum of lytic activity among several well-studied phage groups of proven safety. We propose a modular based approach, utilizing both mono-species and hetero-species phage mixtures. With an approach involving the visual recognition of characteristics exhibited by phages of well studied phage groups on lawns of the standard P. aeruginosa PAO1 strain, the simple and rapid enhancement of the lytic spectrum of cocktails is permitted, allowing the development of tailored preparations for patients capable of circumventing problems associated with phage resistant bacterial mutants.