BACKGROUND

Previous studies with cystic fibrosis transmembrane conductance regulator (CFTR) DeltaF508 mice indicate that estrogen levels may play a role in the occurrence or severity of CF-associated liver disease. However, the underlying mechanisms of liver disease in CF are poorly understood.

METHODS

The levels of SULT1E1 (estrogen sulfotransferase) were measured in livers of control and CFTR-knockout (KO) mice. The impact of increased SULT1E1 activity on hepatic protein expression was assessed by immunoblot and MALDI mass spectrometric analysis.

RESULTS

SULT1E1 expression was significantly elevated in livers of several CFTR-KO mice. SULT1E1 and CFTR were specifically detected in hepatocytes and cholangiocytes, respectively. Elevated SULT1E1 activity may result in lower levels of free beta-estradiol thereby altering estrogen-responsive hepatic protein expression. Estrogen receptors (ER)-alpha and beta were differentially regulated in CFTR-KO and CFTR-DeltaF508 mice. ERalpha expression was reduced in mice with high SULT1E1 activity. Glutathione S-transferase-P1 and carbonic anhydrase III were significantly decreased in CFTR (-/-) mice with high SULT1E1 activity. Furthermore, cytochrome P450 2B9, also estrogen regulated, was significantly induced in the livers of CFTR (-/-) mice with high SULT1E1 activity.

CONCLUSIONS

Elevated SULT1E1 levels and associated alterations in estrogen-regulated hepatic protein expression may play an important role in CF liver disease.

Cystic fibrosis (CF) is a recessive genetic disease characterized by chronic respiratory infections and inflammation causing permanent lung damage. Recurrent infections are caused by Gram-negative antibiotic-resistant bacterial pathogens such as Pseudomonas aeruginosa, Burkholderia cepacia complex (Bcc) and the emerging pathogen genus Pandoraea. In this study, the interactions between co-colonizing CF pathogens were investigated. Both Pandoraea and Bcc elicited potent pro-inflammatory responses that were significantly greater than Ps. aeruginosa. The original aim was to examine whether combinations of pro-inflammatory pathogens would further exacerbate inflammation. In contrast, when these pathogens were colonized in the presence of Ps. aeruginosa the pro-inflammatory response was significantly decreased. Real-time PCR quantification of bacterial DNA from mixed cultures indicated that Ps. aeruginosa significantly inhibited the growth of Burkholderia multivorans, Burkholderia cenocepacia, Pandoraea pulmonicola and Pandoraea apista, which may be a factor in its dominance as a colonizer of CF patients. Ps. aeruginosa cell-free supernatant also suppressed growth of these pathogens, indicating that inhibition was innate rather than a response to the presence of a competitor. Screening of a Ps. aeruginosa mutant library highlighted a role for quorum sensing and pyoverdine biosynthesis genes in the inhibition of B. cenocepacia. Pyoverdine was confirmed to contribute to the inhibition of B. cenocepacia strain J2315. B. multivorans was the only species that could significantly inhibit Ps. aeruginosa growth. B. multivorans also inhibited B. cenocepacia and Pa. apista. In conclusion, both Ps. aeruginosa and B. multivorans are capable of suppressing growth and virulence of co-colonizing CF pathogens.

Cardiac fibrosis caused by adverse cardiac remodeling following myocardial infarction can eventually lead to heart failure. Although the role of soluble factors such as TGF-<em>β</em> is well studied in cardiac fibrosis following myocardial injury, the physiological role of mechanotransduction is not fully understood. Here, we investigated the molecular mechanism and functional role of TRPV4 mechanotransduction in cardiac fibrosis. TRPV4KO mice, 8 weeks following myocardial infarction (MI), exhibited preserved cardiac function compared to WT mice. Histological analysis demonstrated reduced cardiac fibrosis in TRPV4KO mice. We found that WT <em>CF</em> exhibited hypotonicity-induced calcium influx and extracellular matrix (ECM)-stiffness-dependent differentiation in response to TGF-<em>β</em>1. In contrast, TRPV4KO <em>CF</em> did not display hypotonicity-induced calcium influx and failed to differentiate on high-stiffness ECM gels even in the presence of saturating amounts of TGF-<em>β</em>1. Mechanistically, TRPV4 mediated cardiac fibrotic gene promoter activity and fibroblast differentiation through the activation of the Rho/Rho kinase pathway and the mechanosensitive transcription factor MRTF-A. Our findings suggest that genetic deletion of TRPV4 channels protects heart from adverse cardiac remodeling following MI by modulating Rho/MRTF-A pathway-mediated cardiac fibroblast differentiation and cardiac fibrosis.

Deguelin [(7aS,BaS)-13,13a-dihydro-9,10-dimethoxy-3,3-dimethyl-3H-Bis[1]benzopyrano[3,4-b:6',5'-e]pyran-7(7aH)-one], a naturally occurring rotenone, has shown chemopreventive efficacy in several in vivo and in vitro models. In this report, the effectiveness of deguelin at inhibiting the development of AOM-induced colonic ACF was investigated in CF-1 mice. Loss of hex activity was assessed as a second biomarker. In an initial experiment, animals were given s.c. injections of AOM (10 mg/kg body weight) once a week for 2 weeks to induce ACF. Deguelin and vehicle (corn oil) were administered i.g. 7 days a week. Treatment was initiated 2 weeks prior to the first dose of carcinogen and continued for the duration of the study. The mean number of ACF for the control group was 29.0 +/- 4.3, whereas the mean numbers of ACF in the deguelin groups were 24.8 +/- 2.7, 7.2 +/- 1.5 and 4.6 +/- 1.4 at doses of 2.5, 5.0 and 10.0 mg/kg body weight, respectively. In a similar manner, treatment with deguelin significantly (p < 0.001) suppressed the appearance of hex(-) crypts in a dose-dependent manner. In a second study, the ability of deguelin to block the initiation and promotion stages of colon carcinogenesis was investigated. Greatest inhibition was observed when deguelin was administered during the promotional stage (73.3%, p < 0.001). These results demonstrate that deguelin is an efficacious chemopreventive agent against colon carcinogenesis.

Detrimental and beneficial interactions between co-colonizing bacteria may influence the course of infections. In cystic fibrosis (CF) airways, Staphylococcus aureus prevails in childhood, whereas Pseudomonas aeruginosa progressively predominates thereafter. While a range of interactions has been identified, it is unclear if these represent specific adaptations or correlated responses to other aspects of the environment. Here, we investigate how P. aeruginosa adapts to S. aureus by evolving P. aeruginosa in the presence and absence of S. aureus. P. aeruginosa populations that evolved for 150 generations were sequenced and compared to the ancestor strain. Mutations in the Wsp signaling system were identified in both treatments and likely occurred because of low oxygen availability. Despite showing increased killing activity, wsp mutants were less fit in the presence of S. aureus. In contrast, mutations in lipopolysaccharide (LPS) biosynthesis occurred exclusively in co-cultures with S. aureus and conferred a fitness gain in its presence. Moreover, they increased resistance towards beta-lactam antibiotics. Strikingly, both mutations in wsp and LPS genes are observed in clinical isolates from CF-patients. Our results suggest that P. aeruginosa LPS mutations are a direct consequence of S. aureus imposed selection in vitro.

BACKGROUND

Infection with Burkholderia cepacia complex (BCC) is a life threatening complication of cystic fibrosis (CF), often seen as a contraindication for lung transplantation.

METHODS

A long term retrospective study was conducted of all patients with CF undergoing lung transplants from January 1990 to October 2006 in two French centres allowing transplantation in patients colonised with BCC.

RESULTS

22 of the 247 lung transplant patients with CF were infected with BCC (B. cenocepacia genomovar III (n = 8), B. multivorans genomovar II (n = 11), B. vietnamiensis genomovar V (n = 2) and B. stabilis genomovar IV (n = 1)). BCC colonisation was not associated with any significant excess mortality (HR 1.5, 95% CI 0.7 to 3.2; p = 0.58). However, early mortality rates tended to be higher in the BCC group than in the non-BCC group (3 month survival: 85% vs 95%, respectively; log rank p = 0.05). Univariate analysis showed that the risk of death was significantly higher for the eight patients infected with B. cenocepacia than for the other 14 colonised patients (HR 3.2, 95% CI 1.1 to 5.9; p = 0.04). None of the other risk factors tested-primary graft failure, late extubation, septicaemia-had a significant effect. The 5 year cumulative incidence rate of bronchiolitis obliterans syndrome was not significantly higher in the BCC group than in the non-BCC group (38% vs 24%, respectively; p = 0.35).

CONCLUSIONS

Our results suggest that BCC infection with a non-genomovar III organism may not be associated with excess mortality after lung transplantation in patients with CF and should not be seen as sufficient reason to exclude lung transplantation. However, colonisation with B. cenocepacia remains potentially detrimental.

The establishment of set points for cellular activities is essential in regulating homeostasis. Here, we demonstrate key determinants of the fibrogenic set point of cardiac fibroblasts (CFs) by focusing on the pro-fibrotic activity of ATP, which is released by CFs. We tested the hypothesis that the hydrolysis of extracellular ATP by ectonucleoside triphosphate diphosphohydrolases (ENTPDs) regulates pro-fibrotic nucleotide signaling. We detected two ENTPD isoforms, ENTPD-1 and -2, in adult rat ventricular CFs. Partial knockdown of ENTPD-1 and -2 with siRNA increased basal extracellular ATP concentration and enhanced the pro-fibrotic effect of ATP stimulation. Sodium polyoxotungstate-1, an ENTPD inhibitor, not only enhanced the pro-fibrotic effects of exogenously added ATP but also increased basal expression of α-smooth muscle actin, plasminogen activator inhibitor-1 and transforming growth factor (TGF)-β, collagen synthesis, and gel contraction. Furthermore, we found that adenosine, a product of ATP hydrolysis by ENTPD, acts via A2B receptors to counterbalance the pro-fibrotic response to ATP. Removal of extracellular adenosine or inhibition of A2B receptors enhanced pro-fibrotic ATP signaling. Together, these results demonstrate the contribution of basally released ATP in establishing the set point for fibrotic activity in adult rat CFs and identify a key role for the modulation of this activity by hydrolysis of released ATP by ENTPDs. These findings also imply that cellular homeostasis and fibrotic response involve the integration of signaling that is pro-fibrotic by ATP and anti-fibrotic by adenosine and that is regulated by ENTPDs.

Chronic pulmonary infection with Pseudomonas aeruginosa is a feature of cystic fibrosis (CF) and other chronic lung diseases. Cytokines of the interleukin-17 (IL-17) family have been proposed as important in the host response to P. aeruginosa infection through their role in augmenting antibacterial immune responses, although their proinflammatory effect may contribute to lung damage that occurs as a result of chronic infection. We set out to explore the role of IL-17 in the host response to chronic P. aeruginosa infection. We used a murine model of chronic pulmonary infection with CF-related strains of P. aeruginosa We demonstrate that IL-17 cytokine signaling is essential for mouse survival and prevention of chronic infection at 2 weeks postinoculation using two different P. aeruginosa strains. Following infection, there was a marked expansion of cells within mediastinal lymph nodes, comprised mainly of innate lymphoid cells (ILCs); ∼90% of IL-17-producing (IL-17+) cells had markers consistent with group 3 ILCs. A smaller percentage of IL-17+ cells had markers consistent with a BB cell-deficient mice showed that B cell production of IL-17 or natural antibodies did not provide any defense against chronic P. aeruginosa infection. Thus, IL-17 rather than antibody is a key element in host defense against chronic pulmonary infection with P. aeruginosa.

BACKGROUND

A new software tool, Optifood, developed by the WHO and based on linear programming (LP) analysis, has been developed to formulate food-based recommendations.

OBJECTIVE

This study discusses the use of Optifood for predicting whether formulated complementary food (CF) products can ensure dietary adequacy for target populations in Cambodia.

METHODS

Dietary data were collected by 24-h recall in a cross-sectional survey of 6- to 11-mo-old infants (n = 78). LP model parameters were derived from these data, including a list of foods, median serving sizes, and dietary patterns. Five series of LP analyses were carried out to model the target population's baseline diet and 4 formulated CF products [WinFood (WF), WinFood-Lite (WF-L), Corn-Soy-Blend Plus (CSB+), and Corn-Soy-Blend Plus Plus (CSB++)], which were added to the diet in portions of 33 g/d dry weight (DW) for infants aged 6-8 mo and 40 g/d DW for infants aged 9-11 mo. In each series of analyses, the nutritionally optimal diet and theoretical range, in diet nutrient contents, were determined.

RESULTS

The LP analysis showed that baseline diets could not achieve the Recommended Nutrient Intake (RNI) for thiamin, riboflavin, niacin, folate, vitamin B-12, calcium, iron, and zinc (range: 14-91% of RNI in the optimal diets) and that none of the formulated CF products could cover the nutrient gaps for thiamin, niacin, iron, and folate (range: 22-86% of the RNI). Iron was the key limiting nutrient, for all modeled diets, achieving a maximum of only 48% of the RNI when CSB++ was included in the diet. Only WF and WF-L filled the nutrient gap for calcium. WF-L, CSB+, and CSB++ filled the nutrient gap for zinc (9- to 11-mo-olds).

CONCLUSIONS

The formulated CF products improved the nutrient adequacy of complementary feeding diets but could not entirely cover the nutrient gaps. These results emphasize the value of using LP to evaluate special CF products during the intervention planning phase. The WF study was registered at controlled-trials.com as ISRCTN19918531.

OBJECTIVE

Stressors and emotional distress responses impact chronic fatigue syndrome (CFS) symptoms, including fatigue. Having better stress management skills might mitigate fatigue by decreasing emotional distress. Because CFS patients comprise a heterogeneous population, we hypothesized that the role of stress management skills in decreasing fatigue may be most pronounced in the subgroup manifesting the greatest neuroimmune dysfunction.

METHODS

In total, 117 individuals with CFS provided blood and saliva samples, and self-report measures of emotional distress, perceived stress management skills (PSMS), and fatigue. Plasma interleukin-1-beta (IL-1β, IL-2, IL-6, IL-10, and tumor necrosis factor-alpha (TNF-α), and diurnal salivary cortisol were analyzed. We examined relations among PSMS, emotional distress, and fatigue in CFS patients who did and did not evidence neuroimmune abnormalities.

RESULTS

Having greater PSMS related to less fatigue (p=.019) and emotional distress (p<.001), greater diurnal cortisol slope (p=.023) and lower IL-2 levels (p=.043). PSMS and emotional distress related to fatigue levels most strongly in CFS patients in the top tercile of IL-6, and emotional distress mediated the relationship between PSMS and fatigue most strongly in patients with the greatest circulating levels of IL-6 and a greater inflammatory (IL-6):anti-inflammatory (IL-10) cytokine ratio.

CONCLUSIONS

CFS patients having greater PSMS show less emotional distress and fatigue, and the influence of stress management skills on distress and fatigue appear greatest among patients who have elevated IL-6 levels. These findings support the need for research examining the impact of stress management interventions in subgroups of CFS patients showing neuroimmune dysfunction.

The purpose of this study was to compare resistance and cross-resistance development in Pseudomonas aeruginosa isolates from cystic fibrosis (CF) patients to commonly used antipseudomonal antibiotics. Isolates were repeatedly exposed to subinhibitory concentrations of either azlocillin, tobramycin, ceftazidime or ciprofloxacin. On 10 consecutive occasions, samples were removed from the half-MIC well of a microtitre plate and regrown in drug-free medium to provide the next inoculum for MIC determination. The increase in MIC at the end of the treatment period was significant (p<0.05) for all selecting antibiotics. Cross-resistance to unrelated antibiotics was not observed, but was significant (p<0.05) in all beta-lactams (ticarcillin, piperacillin, ceftazidime and cefsulodin) studied where azlocillin was the selecting antibiotic. The addition of clavulanic acid to ticarcillin and of tazobactam to piperacillin had no effect on cross-resistance. The development of resistance to azlocillin was associated with increased beta-lactamase activity and a change in isoelectric point of the beta-lactamases. The result of this study supports a rotational policy for antipseudomonal antibiotics in CF patients.

The aim of this study was to examine the development of resistance of biofilm-growing P. aeruginosa during treatment with ceftazidime. Biofilms were established in vitro using a modified Robbins device (MRD) and in vivo in the rat model of chronic lung infection. Three P. aeruginosa strains isolated from the lungs of cystic fibrosis (CF) patients (MICceftazidime-basal/induced beta-lactamase activity: PAO 579= 0.8 mg/l-19/550 milliunits, 19676A=50 mg/l-38/957 milliunits, 17107B=100 mg/l-504/947 milliunits) were studied. After 1 or 2 weeks of continuous or intermittent (4 h/day) administration of ceftazidime to biofilms established in MDR, a statistically significant development of resistance to ceftazidime in PAO 579 or 19676A bacterial populations occurred. When ceftazidime was administered 4 h/day (200 mg/l) for 2 weeks, the frequency of resistant 19676A having MIC>25 mg/l was 4.4 10(-1) compared to 6.0-10(-5) in the control biofilm. The same trend was observed after continuous administration of ceftazidime. MICceftazidime of the more resistant variants was increased 500-fold for PAO 579 and 8-fold for 19676A, and the specific basal beta-lactamase activities from 19 to 1,400 units for PAO 579 and from 38 to 10,000 units for 19676A. Chronic P. aeruginosa lung infection was established with alginate-embedded PAO 579, 19676A or 17107B in 146 Lewis rats which were treated with ceftazidime 4 g/kg intraperitoneally twice a day for 1 week. A statistically significant development of resistance was observed for all three strains. The MICceftazidime of the more resistant variants was increased 15-fold for PAO 579, 8-fold for 19676A, and 4-fold for 17107B, and the specific basal 3-lactamase activity from 19 to 100 units for PAO 579, from 38 to 1,300 units for 19676A, and from 500 to 1,300 units for 17107B. It was shown that, during treatment with ceftazidime, biofilm-growing P. aeruginosa had the capacity to develop resistance due to the production of chromosomal beta-lactamase.

A recent case of iatrogenic Cushing's syndrome and complete suppression of the pituitary-adrenal-axis in a patient with cystic fibrosis (CF) and allergic bronchopulmonary aspergillosis treated with itraconazole as an antifungal agent, and budesonide as an anti-inflammatory agent led to a systematic assessment of this axis and gonadal function in all patients treated with itraconazole in the authors' CF centre. Itraconazole can inhibit CYP3A, thus interfering with synthesis of gluco- and mineralocorticoids, androgens and oestradiol as well as the metabolism of budesonide. The aim of this study was to evaluate adrenal and gonadal function in patients treated with itraconazole with or without budesonide. An adrenocorticotrophic hormone (ACTH) test (250 microg tetracosactid) was performed in 25 CF patients treated with both itraconazole and budesonide, and in 12 patients treated with itraconazole alone (six patients with CF and six with chronic granulomateous disease). Mineralocorticoid and gonadal steroid function were evaluated by measurements of plasma-renin, follicle stimulating hormone, luteinising hormone, progesterone, oestradiol, testosterone, serum-inhibin A and B. ACTH tests performed as part of a pretransplantation programme in an additional 30 CF patients were used as controls. Eleven of the 25 patients treated with both itraconazole and budesonide had adrenal insufficiency. None of the patients on itraconazole therapy alone nor the control CF patients had a pathological ACTH test. Mineralocorticoid and gonadal insufficiency was not observed in any of the patients. Only one patient with an initial pathological ACTH-test subsequently normalised, the other 10 patients improved but had not achieved normalised adrenal function 2-10 months after itraconazole treatment had been discontinued. Suppression of the adrenal glucocorticoid synthesis was observed in 11 of 25 cystic fibrosis patients treated with both itraconazole and budesonide. The pathogenesis is most likely an itraconazole caused increase in systemic budesonide concentration through a reduced/inhibited metabolism leading to inhibition of adrenocorticotrophic hormone secretion along with a direct inhibition of steroidogenesis. In patients treated with this combination, screening for adrenal insufficiency at regular intervals is suggested.

Cystic fibrosis-related (CF-related) diabetes (CFRD) is an increasingly common and devastating comorbidity of CF, affecting approximately 35% of adults with CF. However, the underlying causes of CFRD are unclear. Here, we examined cystic fibrosis transmembrane conductance regulator (CFTR) islet expression and whether the CFTR participates in islet endocrine cell function using murine models of β cell CFTR deletion and normal and CF human pancreas and islets. Specific deletion of CFTR from murine β cells did not affect β cell function. In human islets, CFTR mRNA was minimally expressed, and CFTR protein and electrical activity were not detected. Isolated CF/CFRD islets demonstrated appropriate insulin and glucagon secretion, with few changes in key islet-regulatory transcripts. Furthermore, approximately 65% of β cell area was lost in CF donors, compounded by pancreatic remodeling and immune infiltration of the islet. These results indicate that CFRD is caused by β cell loss and intraislet inflammation in the setting of a complex pleiotropic disease and not by intrinsic islet dysfunction from CFTR mutation.

Cardiac fibrosis describes the inappropriate proliferation of cardiac fibroblasts (CFs), leading to accumulation of extracellular matrix (ECM) proteins in the cardiac muscle, which is found in many pathophysiological heart conditions. A range of molecular components and cellular pathways, have been implicated in its pathogenesis. In this review, we focus on the TGF-β and WNT signaling pathways, and their mutual interaction, which have emerged as important factors involved in cardiac pathophysiology. The molecular and cellular processes involved in the initiation and progression of cardiac fibrosis are summarized. We focus on TGF-β and WNT signaling in cardiac fibrosis, ECM production, and myofibroblast transformation. Non-coding RNAs (ncRNAs) are one of the main players in the regulation of multiple pathways and cellular processes. MicroRNAs, long non-coding RNAs, and circular long non-coding RNAs can all interact with the TGF-β/WNT signaling axis to affect cardiac fibrosis. A better understanding of these processes may lead to new approaches for diagnosis and treatment of many cardiac conditions. Video Abstract.

Keywords: Cardiac fibrosis; Non-coding RNAs; TGF-β/WNT signaling.

Agricultural use of antimicrobials in subtherapeutic concentrations is increasing in response to the rising demand for food animal products worldwide. In India, the use of antimicrobials in food animal production is unregulated. Research suggests that many clinically important antimicrobials are used indiscriminately. This is the largest study to date in India that surveys poultry production to test for antimicrobial resistance and the occurrence of extended-spectrum β-lactamases (ESBLs) modulated by farming and managerial practices.

Our goal was to survey poultry production for resistance to eleven clinically relevant antimicrobials and phenotypic occurrence of ESBLs as modulated by farming and managerial practices.

Eighteen poultry farms from Punjab were surveyed, and 1,556 Escherichia coli isolates from 530 birds were tested for susceptibility to 11 antimicrobials using the disk diffusion method and validated using VITEK 2 (bioMérieux, Marcy-L’Étoile, France). Samples from 510 of these birds were phenotypically tested for ESBL production using the combination disk method and confirmed using VITEK 2. Generalized linear mixed models were used to infer differences in resistance profiles associated with different farming practices and facility types.

Resistance profiles were significantly different between broiler and layer farms. Broiler farms were 2.2 [ampicillin (AMP), p=0.017] to 23 [nalidixic acid (NX), p<0.001] times more likely to harbor resistant E. coli strains than layer farms. Adjusting for farm type (broiler vs. layer), the odds of resistance (although not statistically significant) to all antimicrobials except nitrofurantoin (NIT) were higher in independent facilities (IUs) as compared to contracted facilities (CFs). Increased prevalence of multidrug resistance (MDR; 94% compared to 60% in layers), including prevalence of ESBL-producing strains (87% compared to 42% in layers), was observed in broiler farms.

Our findings suggest that unregulated use of clinically relevant antimicrobials in Indian broiler and layer farms may contribute to the emergence of resistance and support the need to curb the nontherapeutic use of medically important antimicrobials in food animal production. https://doi.org/10.1289/EHP292.

Pseudomonas aeruginosa, a major respiratory pathogen in cystic fibrosis (CF) patients, facilitates infection by other opportunistic pathogens. Burkholderia cenocepacia, which normally infects adolescent patients, encounters alginate elaborated by mucoid P. aeruginosa. To determine whether P. aeruginosa alginate facilitates B. cenocepacia infection in mice, cystic fibrosis transmembrane conductance regulator knockout mice were infected with B. cenocepacia strain BC7 suspended in either phosphate-buffered saline (BC7/PBS) or P. aeruginosa alginate (BC7/alginate), and the pulmonary bacterial load and inflammation were monitored. Mice infected with BC7/PBS cleared all of the bacteria within 3 days, and inflammation was resolved by day 5. In contrast, mice infected with BC7/alginate showed persistence of bacteria and increased cytokine levels for up to 7 days. Histological examination of the lungs indicated that there was moderate to severe inflammation and pneumonic consolidation in isolated areas at 5 and 7 days postinfection in the BC7/alginate group. Further, alginate decreased phagocytosis of B. cenocepacia by professional phagocytes both in vivo and in vitro. P. aeruginosa alginate also reduced the proinflammatory responses of CF airway epithelial cells and alveolar macrophages to B. cenocepacia infection. The observed effects are specific to P. aeruginosa alginate, because enzymatically degraded alginate or other polyuronic acids did not facilitate bacterial persistence. These observations suggest that P. aeruginosa alginate may facilitate B. cenocepacia infection by interfering with host innate defense mechanisms.

OBJECTIVE

To examine the possible relationship between maternal and fetal characteristics and pregnancy outcomes on fetal and maternal cell-free (cf) DNA in maternal plasma at 11-13 weeks' gestation.

METHODS

cfDNA was extracted from maternal plasma of 1,949 singleton pregnancies and chromosome-selective sequencing was used to determine the proportion of cfDNA and total cfDNA counts which was of fetal and maternal origin. Multivariate regression analysis was used to determine whether specific maternal and fetal characteristics and pregnancy complications, such as preeclampsia (PE), early spontaneous preterm birth (SPB) and delivery of small for gestational age (SGA) neonates, were significant predictors of fetal and maternal cfDNA in maternal plasma.

RESULTS

The fetal and maternal cfDNA plasma concentration increased with serum pregnancy-associated plasma protein-A and free β-human chorionic gonadotropin level, was higher in women of Afro-Caribbean and East-Asian racial origin than in Caucasians, and lower in smokers, but it was not significantly altered in pregnancies complicated by PE, SPB or SGA. The fetal cfDNA level was inversely related to maternal weight and uterine artery pulsatility index, and maternal cfDNA increased with maternal weight.

CONCLUSIONS

The fetal and maternal cfDNA level in maternal plasma is affected by maternal and fetal characteristics, but it is not altered in pregnancy complications.

The bacterial community inhabiting the water column at Terra Nova Bay (Ross Sea, Antarctica) was examined by the fluorescent in situ hybridization (FISH) technique and the genotypic and phenotypic characterization of 606 bacterial isolates. Overall, the FISH analysis revealed a bacterioplankton composition that was typical of Antarctic marine environments with the Cytophaga/Flavobacter (CF) group of Bacteroidetes that was equally dominant with the Actinobacteria and Gammaproteobacteria. As sampling was performed during the decay of sea-ice, it is plausible to assume the origin of Bacteroidetes from the sea-ice compartment where they probably thrive in high concentration of DOM which is efficiently remineralized to inorganic nutrients. This finding was supported by the isolation of Gelidibacter, Polaribacter, and Psychroflexus members (generally well represented in Antarctic sea-ice) which showed the ability to hydrolyze macromolecules, probably through the production of extracellular enzymes. A consistently pronounced abundance of the Gammaproteobacteria (67.8%) was also detected within the cultivable fraction. Altogether, the genera Psychromonas and Pseudoalteromonas accounted for 65.4% of total isolates and were ubiquitous, thus suggesting that they may play a key role within the analyzed bacterioplankton community. In particular, Pseudoalteromonas isolates possessed nitrate reductase and were able to hydrolyze substrates for protease, esterase, and β-galactosidase, thus indicating their involvement in the carbon and nitrogen cycling. Finally, the obtained results highlight the ability of the Actinobacteria to survive and proliferate in the Terra Nova Bay seawater as they generally showed a wide range of salt tolerance and appeared to be particularly competitive with strictly marine bacteria by better utilizing supplied carbon sources.

<A<em>b</em>stractText>Ivacaftor is the first in a class of drugs, <em>CF</em>TR modulators, that target the underlying defect in cystic fi<em>b</em>rosis (<em>CF</em>). This long-term o<em>b</em>servational safety study evaluated <em>CF</em> disease progression in patients treated with ivacaftor in a real-world setting for up to 5 years.</A<em>b</em>stractText><A<em>b</em>stractText>Data from existing US and UK <em>CF</em> patient registries were used to assess longitudinal patterns in lung function, nutritional status, pulmonary exacer<em>b</em>ations and hospitalizations, <em>CF</em>-related dia<em>b</em>etes (<em>CF</em>RD), and Pseudomonas aeruginosa in ivacaftor-treated vs untreated comparator cohorts matched <em>b</em>y age, sex, and disease severity.</A<em>b</em>stractText><p><div>(<em>b</em>)RESULTS</<em>b</em>)</div>US analyses included 635 ivacaftor-treated patients and 1874 comparators followed for 5 years from year 1 of market availa<em>b</em>ility (2012-2016). Evaluation of outcome patterns from pretreatment <em>b</em>aseline (2011) through year 5 (2016), showed that relative to comparators, ivacaftor-treated patients had <em>b</em>etter preserved lung function (mean change in percent predicted FEV<su<em>b</em>)1</su<em>b</em>), -0.7 percentage points with ivacaftor vs -8.3 percentage points in comparators) and improved nutritional status (mean <em>b</em>ody mass index change +2.4 kg/m² with ivacaftor vs +1.6 kg/m² in comparators). US patients treated with ivacaftor had significantly lower frequencies of exacer<em>b</em>ations and hospitalizations in each of the 5 years of follow-up relative to pretreatment <em>b</em>aseline and comparators. Favora<em>b</em>le trends in <em>CF</em>RD and P. aeruginosa prevalence were also o<em>b</em>served. Findings from the smaller UK registry were directionally similar to and consistent with US findings.</p><A<em>b</em>stractText>This o<em>b</em>servational study represents the largest longitudinal analysis of patients treated with ivacaftor in a real-world setting. The findings support disease modification <em>b</em>y <em>CF</em>TR modulation with ivacaftor.</A<em>b</em>stractText>

(<em>b</em>)Rationale:</<em>b</em>) Enhancing non-<em>CF</em>TR mediated anion secretion is an attractive therapeutic approach for the treatment of cystic fi<em>b</em>rosis and other muco-o<em>b</em>structive diseases. (<em>b</em>)O<em>b</em>jectives:</<em>b</em>) To determine the effects of TMEM16A potentiation upon epithelial fluid secretion and mucociliary clearance. (<em>b</em>)Methods:</<em>b</em>) The effects of a novel low molecular weight TMEM16A potentiator (ETX001) were evaluated in human cell and animal models of airway epithelial function and mucus transport. (<em>b</em>)Measurements & Main Results:</<em>b</em>) Potentiating the activity of TMEM16A with ETX001 increased the Ca2+-activated Cl- channel activity and anion secretion in human <em>b</em>ronchial epithelial cells from cystic fi<em>b</em>rosis patients without impacting on calcium signalling. ETX001 rapidly increased fluid secretion and airway surface liquid height in cystic fi<em>b</em>rosis human <em>b</em>ronchial epithelial cells under <em>b</em>oth static and conditions designed to mimic the shear stress associated with tidal <em>b</em>reathing. In ovine models of mucus clearance (tracheal mucus velocity and mucociliary clearance), inhaled ETX001 was a<em>b</em>le to accelerate clearance <em>b</em>oth when <em>CF</em>TR function was reduced <em>b</em>y administration of a pharmacological <em>b</em>locker and when <em>CF</em>TR was fully functional. (<em>b</em>)Conclusions:</<em>b</em>) Enhancing the activity of TMEM16A increases epithelial fluid secretion and enhances mucus clearance independent of <em>CF</em>TR function. TMEM16A potentiation is a novel approach for the treatment of patients with cystic fi<em>b</em>rosis and non-<em>CF</em> muco-o<em>b</em>structive diseases. This article is open access and distri<em>b</em>uted under the terms of the Creative Commons Attri<em>b</em>ution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/<em>b</em>y-nc-nd/4.0/).

Rapidly growing, non-tuberculous mycobacteria (NTM) in the Mycobacterium abscessus (MAB) species are emerging pathogens that cause various diseases including skin and respiratory infections. The species has undergone recent taxonomic nomenclature refinement, and is currently recognized as two subspecies, M. abscessus subsp. abscessus (MAB-A) and M. abscessus subsp. bolletii (MAB-B). The recently reported outbreaks of MAB-B in surgical patients in Brazil from 2004 to 2009 and in cystic fibrosis patients in the United Kingdom (UK) in 2006 to 2012 underscore the need to investigate the genetic diversity of clinical MAB strains. To this end, we sequenced the genomes of two Brazilian MAB-B epidemic isolates (CRM-0019 and CRM-0020) derived from an outbreak of skin infections in Rio de Janeiro, two unrelated MAB strains from patients with pulmonary infections in the United States (US) (NJH8 and NJH11) and one type MAB-B strain (CCUG 48898) and compared them to 25 publically available genomes of globally diverse MAB strains. Genome-wide analyses of 27,598 core genome single nucleotide polymorphisms (SNPs) revealed that the two Brazilian derived CRM strains are nearly indistinguishable from one another and are more closely related to UK outbreak isolates infecting CF patients than to strains from the US, Malaysia or France. Comparative genomic analyses of six closely related outbreak strains revealed geographic-specific large-scale insertion/deletion variation that corresponds to bacteriophage insertions and recombination hotspots. Our study integrates new genome sequence data with existing genomic information to explore the global diversity of infectious M. abscessus isolates and to compare clinically relevant outbreak strains from different continents.

Although most species in the genus Burkholderia are not pathogenic for healthy persons, a few are capable of causing severe, life threatening infection. B. mallei and B. pseudomallei are the causative agents of glanders and melioidosis, respectively. Interest in these species has increased recently owing to their potential for use as agents of bioterrorism. B. cepacia emerged during the past two decades as an important opportunistic pathogen among persons with certain underlying diseases. Persons with chronic granulomatous disease, a primary immunodeficiency, or cystic fibrosis (CF), the most common lethal inherited disorder in Caucasians, are at particular risk. In CF, respiratory tract infection may be chronic or associated with a rapid deterioration in pulmonary function. Studies in the early 1990s utilized a variety of genotyping techniques to provide compelling evidence of person-to-person transmission of B. cepacia among CF patients. This prompted the institution of rigorous infection control measures that have placed a heavy burden on persons with CF. More recent work has demonstrated that several distinct bacterial species actually exist among bacteria previously identified merely as B. cepacia. How these species, collectively referred to as the B. cepacia complex, differ with respect to their epidemiology, natural history, and pathology in CF is the subject of ongoing investigation.

To determine if oral dosing with the CFTR-potentiator ivacaftor (VX-770, Kalydeco) improves CFTR-dependent sweating in CF subjects carrying G551D or R117H-5T mutations, we optically measured sweat secretion from 32-143 individually identified glands in each of 8 CF subjects; 6 F508del/G551D, one G551D/R117H-5T, and one I507del/R117H-5T. Two subjects were tested only (-) ivacaftor, 3 only (+) ivacaftor and 3 (+/-) ivacaftor (1-5 tests per condition). The total number of gland measurements was 852 (-) ivacaftor and 906 (+) ivacaftor. A healthy control was tested 4 times (51 glands). For each gland we measured both CFTR-independent (M-sweat) and CFTR-dependent (C-sweat); C-sweat was stimulated with a β-adrenergic cocktail that elevated [cAMP]i while blocking muscarinic receptors. Absent ivacaftor, almost all CF glands produced M-sweat on all tests, but only 1/593 glands produced C-sweat (10 tests, 5 subjects). By contrast, 6/6 subjects (113/342 glands) produced C-sweat in the (+) ivacaftor condition, but with large inter-subject differences; 3-74% of glands responded with C/M sweat ratios 0.04%-2.57% of the average WT ratio of 0.265. Sweat volume losses cause proportionally larger underestimates of CFTR function at lower sweat rates. The losses were reduced by measuring C/M ratios in 12 glands from each subject that had the highest M-sweat rates. Remaining losses were estimated from single channel data and used to correct the C/M ratios, giving estimates of CFTR function (+) ivacaftor = 1.6%-7.7% of the WT average. These estimates are in accord with single channel data and transcript analysis, and suggest that significant clinical benefit can be produced by low levels of CFTR function.