Two hundred thirty-six patients with T3 bladder cancer who survived radical surgery and proved to have P3a, P3b, or P4a tumors were randomized in two phases into three groups: (a) no further treatment (83 patients); (b) postoperative radiotherapy multiple daily fractionation (MDF), using 3 daily fractions of 1.25 Gy each, with 3 hr between fractions, up to a total dose of 37.5 Gy in 12 days (75 patients); and (c) postoperative radiotherapy conventional fractionation (CF), for a total dose of 50 Gy/5 weeks (78 patients). The tolerance of the patients to postoperative radiotherapy was quite acceptable, with equal acute reactions in MDF and CF groups. The 5-year disease-free survival (DFS) rates amounted to 49 and 44% in MDF and CF postoperative radiotherapy groups, respectively, compared to 25% in the cystectomy-alone group. The 5-year local control rates were 87% and 93% for those treated with multiple daily fractionation and conventional fractionation while it was 50% in the surgery-alone group. The therapeutic benefit of postoperative irradiation was consistent for all tumor types, histological grades, and pathological stages for both the disease-free survival and local control. Patients with nodal metastases demonstrated lower recurrence rates in the postoperative radiotherapy groups, but this was not associated with improved disease-free survival. Multivariate analysis using the Cox Model confirmed these results. The independent prognostic factors affecting both disease-free survival and local control were the addition of postoperative radiotherapy, the nodal status, the pathological stage, and the tumor grade. Late complications of radiotherapy in the skin, small intestine, rectum, and the anastomotic site of the urinary division were lower with MDF than with conventional fractionation.

Cystic fibrosis is the most common and best known genetic disease involving a defect in transepithelial Cl- transport by mutations in the CF gene on chromosome 7, which codes for the cystic fibrosis transmembrane conductance regulator protein (CFTR). The most serious symptoms are observed in the lungs, augmenting the risk of bacterial infection. The objective of this review was to describe the bacterial pathogens colonizing patients with cystic fibrosis. A systematic search was conducted using the international bibliographic databanks SCIELO, HIGHWIRE, PUBMED, SCIRUS and LILACS to provide a useful and practical review for healthcare workers to make them aware of these microorganisms. Today, B. cepacia, P. aeruginosa and S. aureus are the most important infectious agents in cystic fibrosis patients. However, healthcare professionals must pay attention to emerging infectious agents in these patients, because they represent a potentially serious future problem. Therefore, these pathogens should be pointed out as a risk to these patients, and hospitals all over the world must be prepared to detect and combat these bacteria.

There is now some evidence that chronic fatigue syndrome is accompanied by an activation of the inflammatory response system and by increased oxidative and nitrosative stress. Nuclear factor kappa beta (NFkappabeta) is the major upstream, intracellular mechanism which regulates inflammatory and oxidative stress mediators. In order to examine the role of NFkappabeta in the pathophysiology of CFS, this study examines the production of NFkappabeta p50 in unstimulated, 10 ng/mL TNF-alpha (tumor necrosis factor alpha) and 50 ng/mL PMA (phorbolmyristate acetate) stimulated peripheral blood lymphocytes of 18 unmedicated patients with CFS and 18 age-sex matched controls. The unstimulated (F=19.4, df=1/34, p=0.0002), TNF-alpha-(F=14.0, df=1/34, p=0.0009) and PMA-(F=7.9, df=1/34, p=0.008) stimulated production of NFkappabeta were significantly higher in CFS patients than in controls. There were significant and positive correlations between the production of NFkappabeta and the severity of illness as measured with the FibroFatigue scale and with symptoms, such as aches and pain, muscular tension, fatigue, irritability, sadness, and the subjective feeling of infection. The results show that an intracellular inflammatory response in the white blood cells plays an important role in the pathophysiology of CFS and that previous findings on increased oxidative stress and inflammation in CFS may be attributed to an increased production of NFkappabeta. The results suggest that the symptoms of CFS, such as fatigue, muscular tension, depressive symptoms and the feeling of infection reflect a genuine inflammatory response in those patients. It is suggested that CFS patients should be treated with antioxidants, which inhibit the production of NFkappabeta, such as curcumin, N-Acetyl-Cysteine, quercitin, silimarin, lipoic acid and omega-3 fatty acids.

In many plant-pathogen interactions resistance to disease is controlled by the interaction of plant-encoded resistance (R) genes and pathogen-encoded avirulence (Avr) genes. The interaction between tomato and the leaf mould pathogen Cladosporium fulvum is an ideal system to study the molecular basis of pathogen perception by plants. A total of four tomato genes for resistance to C. fulvum (Cf-2, Cf-4, Cf-5 and Cf-9) have been isolated from two genetically complex chromosomal loci. Their gene products recognize specific C. fulvum-encoded avirulence gene products (Avr2, Avr4, Avr5 and Avr9) by an unknown molecular mechanism. Cf genes encode extracellular membrane-anchored glycoproteins comprised predominantly of 24 amino acid leucine-rich repeats (LRRs). Cf genes from the same locus encode proteins which are more than 90% identical. Most of the amino-acid sequence differences correspond to the solvent-exposed residues within a beta-strand/beta-turn structural motif which is highly conserved in LRR proteins. Sequence variability within this motif is predicted to affect the specificity of ligand binding. Our analysis of Cf gene loci at the molecular level has shown they comprise tandemly duplicated homologous genes, and suggests a molecular mechanism for the generation of sequence diversity at these loci. Our analysis provides further insight into the molecular basis of pathogen perception by plants and the organization and evolution of R gene loci.

BACKGROUND

Colonisation with Burkholderia cepacia complex pathogens has been associated with accelerated decline in cystic fibrosis (CF) patients. The two most common species among the CF community are Burkholderia cenocepacia and Burkholderia multivorans. However, Burkholderia dolosa has recently been causing concern due to its transmissibility and virulence in CF patients.

METHODS

We have compared the ability of five B. dolosa strains to invade lung epithelial cells in vitro with other members of the Bcc. The bacterial epithelial cell interaction was visualised by transmission electron microscopy. We have also examined the ability of these strains to form biofilms in vitro.

RESULTS

We have found that members of this species can invade pulmonary epithelial cells in vitro as readily as those from B. cenocepacia and B. multivorans. Confirmation of intracellular invasion was obtained by transmission electron microscopy. B. dolosa strains were readily observed in membrane bound vesicles inside the lung epithelial cells. In addition, strains from this species were capable of forming strong biofilms at a level comparable to the more clinically relevant species.

CONCLUSIONS

B. dolosa shows comparable virulence characteristics in vitro to the two most clinically relevant species indicating precautions should be taken when this species is identified in the CF population.

The serum protein cystic fibrosis-associated antigen (CFAG), present at elevated levels in CF homozygotes and heterozygotes, is now known to consist of two distinct but related subunits (calgranulins A (CAGA) and B (CAGB)). Both show similarity to the S100-related calcium-binding proteins. We have previously assigned CAGA to human chromosome 1q12-q21 and demonstrate here that the cDNA probe for CAGB cosegregates with it in our somatic cell hybrid panel. cDNA probes for the related genes calcyclin (CACY) and a mouse placental protein (18A2, suggested name Capl) enabled us to confirm and refine the in situ hybridization result assigning CACY to chromosome 1q21-25 and to demonstrate that both genes cosegregate with CAGA and CAGB. Capl was mapped to a region of chromosome 3 in the mouse using the BXD recombinant inbred strain mice where the p11 protein (calpactin light chain Cal1l), another S100 family member, has been localized. Cacy is shown to be within 8 kb of Capl in the mouse genome.

Although resting hemodynamic load has been extensively investigated as a determinant of left ventricular (LV) hypertrophy, little is known about the relationship between provoked hemodynamic load and the risk of LV hypertrophy. We studied central pressure-flow relations among 40 hypertensive and 19 normotensive adults using carotid applanation tonometry and Doppler echocardiography at rest and during a 40% maximal voluntary forearm contraction (handgrip) maneuver. Carotid-femoral pulse wave velocity (CF-PWV) was measured at rest. Hypertensive subjects demonstrated various abnormalities in resting and induced pulsatile load. Isometric exercise significantly increased systemic vascular resistance, aortic characteristic impedance (Zc), induced earlier wave reflections, increased augmentation index, and decreased total arterial compliance (TAC; all P < or = 0.01). In hypertensive subjects, CF-PWV was the strongest resting predictor of LV mass index (LVMI) and remained an independent predictor after adjustment for age, gender, systemic vascular resistance, reflection magnitude, aortic Zc, and TAC (beta = 2.52 m/s; P < 0.0001). Age, sex, CF-PWV, and resting hemodynamic indexes explained 48% of the interindividual variability in LVMI. In stepwise regression, TAC (beta = -17.85; P < 0.0001) during handgrip, Zc during handgrip (beta = -150; P < 0.0001), and the change in the timing of wave reflections during handgrip (beta = -0.63; P = 0.03) were independent predictors of LVMI. A model that included indexes of provoked hemodynamic load explained 68% of the interindividual variability in LVMI. Hemodynamic load provoked by isometric exercise strongly predicts LVMI in hypertension. The magnitude of this association is far greater than for resting hemodynamic load, suggesting that provoked testing captures important arterial properties that are not apparent at rest and is advantageous to assess dynamic arterial load in hypertension.

Burkholderia cepacia complex (Bcc) is a group of Gram-negative pulmonary pathogens associated with life-threatening infections in patients with cystic fibrosis (CF). The airway epithelium plays a crucial role in the initiation and modulation of inflammatory responses to these pathogens. Interleukin (IL)-8 released from epithelial cells is a potent chemoattractant for neutrophils. The aims of this study were to compare the IL-8 response to Bcc infection in different epithelial cell types and to investigate the impact of IL-8 on Bcc growth and intracellular survival. To compare epithelial cell IL-8 responses, 4 human epithelial cell lines were used in the study; A549 cells, an alveolar epithelial cell line, Calu-3 cells, a sub-bronchial epithelial cell line, 16HBE14o- cells, and CFBE41o- cells, which are CFTR-positive and CFTR-negative bronchial epithelial cell lines, respectively. Two B. multivorans and 2 B. cenocepacia strains all induced a significant IL-8 response by 12 h and further increased in all cell lines at 24 h. Furthermore, the levels of IL-8 from Calu-3 and A549 cells were approximately 3 times that of 16HBE14o- or CFBE41o- cells. In 2 of the cell lines examined (16HBE14o- and CFBE41o-), B. cenocepacia LMG 16656 (J2315), an epidemic strain, induced greater levels of IL-8 (P<0.01) compared to other Bcc strains tested. The CFTR-positive and -negative cell lines secreted similar levels of IL-8 indicating a CFTR-independent induction of IL-8. However, the CFTR-negative cells did secrete constitutive levels of IL-8 greater than that of CFTR-positive cells. An investigation of the effect of IL-8 on Bcc extracellular and intracellular growth found that at low concentrations (<10 ng/ml) of recombinant human (rh) IL-8, the growth of B. cenocepacia LMG 16656 and B. multivorans LMG 13010 was enhanced, whereas at higher concentrations (10 ng/ml), growth of both strains was significantly reduced. Growth of both non-CF Bcc strains remained unchanged in the presence of rhIL-8. In contrast to extracellular growth, higher concentrations (10ng/ml) of rhIL-8 enhance the intracellular growth and survival of both LMG 16656 and LMG 13010 in 16HBE14o- and CFBE41o- cell lines. Although LMG 13010 uptake by epithelial cells was higher than LMG 16656 (P<0.01), the intracellular growth of LMG 16656 is greater than LMG 13010 (P<0.05). These studies demonstrated that the type of epithelial cells encountered by Bcc strains determines the extent of the IL-8 responses triggered and that this cytokine in addition to its well-established proinflammatory properties can enhance both the extracellular and intracellular growth of Bcc strains.

There is a need to judge general exercise tolerance in children with cystic fibrosis (CF) under normal daily activity conditions and -when more extensive testing is required-in an exercise laboratory in a specialized center. We investigated the reproducibility, validity, and criterion for a 6-minute walking test, which simulates normal childhood activities. In Part A, we evaluated the reproducibility of a 6-minute walking test in 23 children (12 girls and 11 boys; ages 11.1 +/- 2.2 years; range, 8.2 15.6 years) with mild symptoms of CF [forced expiratory volume in 1 second (FEV1) 94.4 +/- 16.5% of predicted values (range, 60.6-129.7); body weight Z-score -0.71 +/- 0.81 (range, -1.73-0.93)]. The subjects performed two standardized 6-minute walking tests with 1 week between tests. There was no significant difference between the two walking distances reached (737 +/- 85 versus 742 +/- 90 meters; P = 0.56), and there was a strong correlation between the two walking distances reached by the individuals (r = 0.90, P < 0.0001). In Part B, the validity of the walking test was evaluated in 15 children (6 girls and 9 boys; ages 14.5 +/- 2.0 years; range, 10.2-16.9 years) with moderate symptoms of CF [FEV1 = 58 +/- 16.0% of predicted values, (range, 41.1-89.4); RV/TLC ratio = 46.3 +/- 6.5% (range, 31.6-57.2); body weight Z-score: -1.29 +/- 0.60 (range, -2.20-0.14)]. They underwent standardized maximum incremental exercise testing on a cycle ergometer and a 6-minute walking test. Postexertional lactate values exceeded threshold values (as described in the literature) in all patients but one. Correlation analysis (Pearson) showed a significant correlation between the walking distance reached (WD = 697 +/- 104 meters), and the maximum workload (Wmax = 118 +/- 44 watt; r = 0.76, P < 0.001) or the maximum oxygen uptake (1,688 +/- 495 ml; r = 0.76, P < 0.001), the latter two being determined on a cycle ergometer, RV/TLC% showed a significant negative correlation (r = -0.72, P < 0.01) with WD. Stepwise multiple regression analysis showed a multiple regression coefficient of R = 0.84 (P < 0.001) for Wmax and RV/TLC % as the independent variables vs. WD as the dependent variable. We conclude that the 6-minute walking test is a valid and useful test in children with mild to moderate symptoms of CF to assess their exercise tolerance and endurance. Exercise test results correlated negatively with pulmonary hyperinflation expressed by the RV/TLC ratio.

A highly transmissible strain of Burkholderia cepacia from genomovar III carries the cable pilin gene, expresses the 22 kDa adhesin (cblA +ve/Adh +ve), binds to cytokeratin 13 (CK13) and is invasive. CK13 is expressed abundantly in the airway epithelia of cystic fibrosis (CF) patients. We have now investigated whether binding of cblA +ve/Adh +ve B. cepacia to CK13 potentiates bacterial invasion and epithelial damage using bronchial epithelial cell cultures differentiated into either squamous (CK13-enriched) or mucociliary (CK13-deficient) epithelia. Three different B. cepacia isolates (cblA +ve/Adh +ve, cblA +ve/Adh -ve and cblA -ve/Adh -ve) showed minimal binding to mucociliary cultures, and did not invade or cause cell damage. In contrast, the cblA +ve/Adh +ve isolate, but not others, bound to CK13-expressing cells in squamous cultures, caused cytotoxicity and stimulated IL-8 release within 2 h. By 24 h, this isolate invaded and migrated across the squamous culture, causing moderate to severe epithelial damage. A specific antiadhesin antibody, which blocked the initial binding of the cblA +ve/Adh +ve isolate to CK13, significantly inhibited all the pathologic effects. Transmission electron microscopy of squamous cultures incubated with the cblA +ve/Adh +ve isolate, revealed bacteria on the surface surrounded by filopodia by 2 h, and within the cells in membrane-bound vesicles by 24 h. Bacteria were also observed free in the cytoplasm, surrounded by intermediate filaments containing CK13. These findings suggest that binding of B. cepacia to CK13 is an important initial event and that it promotes bacterial invasion and epithelial damage.

Factor analysis models have played a central role in formulating conceptual models in personality and personality assessment, as well as in empirical examinations of personality measurement instruments. Yet, the use of item-level data presents special problems for factor analysis, applications. In this article, we review recent developments in factor analysis that are appropriate for the type of item-level data often collected in personality. Included in this review are discussions of how these developments have been addressed in the context of two different (but formally related) statistical models item response theory (IRT: Hambleton, Swaminathan, & Rogers, 1991) and structural, equation modeling (Bollen 1989) for item-level data. We also discuss the relevance of item scaling in the context of these models. Using the restandardization data for the Minnesota Multiphasic Personality Inventory-2 Scale (cf. Butcher, Dahlstrom, Graham, Tellegen, & Kaemmer, 1989), we show brief examples of the utility of these approaches to address basic questions about responses to personality scale items regarding: (a) scale, dimensionality and general item properties, (b) the "appropriateness" of the observed responses, and (c) differential item functioning across subsamples. implications for analyses of personality item-level data in the IRT and factor analytic traditions are discussed.

OBJECTIVE

To identify the routes of transmission in a nosocomial outbreak of hepatitis C virus (HCV) infection.

METHODS

Epidemiological investigation, including screening for HCV of hospitalized patients, and a retrospective cohort study, review of hygiene and medical practices, and molecular comparison of HCV isolates.

METHODS

A specialized care unit for cystic fibrosis (CF) and diabetic patients at an acute-care facility in the south of France.

RESULTS

Of the 57 CF patients (age in 1995: 2-28 years), 38 (66.7%) were tested and 22 (57.9%) were anti-HCV positive. Eight (50%) of 16 patients with anti-HCV antibody tested by polymerase chain reaction were viremic. No patients had received blood products or had any history of intravenous drug use. All 18 (100%) patients with CF who had ever undergone self-monitoring of capillary blood glucose in the unit were anti-HCV positive, compared to 4 (20%) of 20 who had not (relative risk, 5.0; 95% confidence interval, 2.1-12.0). Seventy (39.5%) of the patients with diabetes were screened for anti-HCV; 12 (18.8%) tested positive, with 3 (25%) positive for HCV-RNA. Patients with diabetes had routine capillary blood glucose monitoring while hospitalized and shared with CF patients the same spring-triggered devices for capillary blood glucose monitoring. The disposable platform of the devices was not changed between patient use. All HCV isolates belonged to the type 1, subtype b, and phylogenetic analysis showed a close homology by sequencing of NS5b and E2/HVR regions.

CONCLUSIONS

As reported earlier for the hepatitis B virus, shared spring-triggered devices for capillary blood glucose monitoring by finger puncture may transmit HCV. Strict application of Standard Precautions procedures is warranted in any healthcare setting.

Adenoviral vectors have been used successfully to transfer the human CFTR cDNA to respiratory epithelium in animal models and to CF patients in vivo. However, studies done primarily in mice, indicate that present vector systems have limitations. Among other things, transgene expression in the lung is transient and the production of neutralizing antibodies against adenovirus correlates with a reduced ability to readminister a vector of the same serotype. Here we demonstrate that in mice, a transient blockade of costimulation between activated T cells and B cells/antigen presenting cells using a monoclonal antibody (MR1) against murine CD40 ligand inhibits the development of neutralizing antibodies to adenoviral (Ad) vector. MR1 also decreased the cellular immune response to Ad vector and allowed an increase in persistence of transgene expression. Furthermore, when administered with a second dose of Ad vector to mice preimmunized against vector, MR1 was able to interfere with the development of a secondary antibody response and allowed for high levels of transgene expression upon a third administration of vector to the airway.

OBJECTIVE

To investigate in vitro if P-glycoprotein (P-gp) transport can differentiate between antibiotic drugs exhibiting increased active renal clearance (CL(r)) in cystic fibrosis (CF) patients (i.e., dicloxacillin, trimethoprim) and drugs that do not exhibit this phenomenon (i.e.. cefsulodin, sulfamethoxazole).

METHODS

Transport studies were carried out in MDCK (wild type) and MDR1-MDCK (P-gp overexpressing) cells that were grown to confluence on Transwell inserts. [14C]-mannitol transport and transepithelial electrical resistance (TEER) were measured to validate the integrity of the cells. Drug concentrations were analyzed using HPLC.

RESULTS

Dicloxacillin and trimethoprim are substrates of P-gp (B->>A/A->>B ratios in MDR1-MDCK cells are 32 and 50, respectively). P-gp inhibitors (i.e., GG918, cyclosporine, ketoconazole, vinblastine) decreased the B->>A transport of dicloxacillin and trimethoprim and increased the A->>B transport of trimethoprim while non-P-gp inhibitors (e.g., PAH) had no effect. In contrast, cefsulodin and sulfamethoxazole are not substrates of P-gp (B->>sA/A->>B values in MDCK and MDR1-MDCK cells are -1).

CONCLUSIONS

Our in vitro studies suggest that P-glycoprotein may play a role in increasing renal clearance of drug substrates in CF patients. Dicloxacillin and trimethoprim. which are both substrates of P-gp, show increased active renal clearance in CF patients while cefsulodin and sulfamethoxazole, which are not P-gp substrates, do not show increased active renal clearance in CF patients.

To explore the physiology of cholecystokinin (CCK) in humans, we investigated the effect on gallbladder contraction and gastric emptying of a recently developed CCK receptor antagonist, MK-329. In a double-blind, four-period crossover study eight subjects received single doses of 0.5, 2, or 10 mg MK-329, or placebo, followed by an intravenous infusion of CCK-8 (30 pmol/kg.h). In placebo-treated subjects gallbladder volumes decreased on average to 43% of initial volumes after 2 h of CCK infusion. MK-329 caused a dose-dependent inhibition of CCK-stimulated gallbladder contraction with 10 mg producing complete blockade (P less than 0.01, cf. placebo). Gallbladder contraction and gastric emptying rates after a mixed meal were then measured in a two-period crossover study. Subjects received placebo or 10 mg of MK-329 2 h before eating. Gastric emptying of both solids and liquids was measured simultaneously by gamma scintigraphy. In placebo-treated subjects plasma CCK levels increased postprandially to 2.3 pM, gallbladder volumes decreased 68.4 +/- 3.8% (SE), and the times for 50% emptying of liquids and solids from the stomach were 58 +/- 10 and 128 +/- 8 min, respectively. In MK-329-treated subjects there was a marked elevation in peak CCK levels to 13.8 pM (P less than 0.01, cf. placebo), and gallbladder contraction was completely inhibited. Solid and liquid emptying rates were unaffected. These findings demonstrate that (a) MK-329 is a potent, orally active antagonist of CCK in humans, and (b) CCK is the major regulator of postprandial gallbladder contraction. These data also support the concept of negative feedback regulation of CCK secretion and suggest that mechanisms other than CCK play a dominant role in the regulation of postprandial gastric emptying rates.

Human beta-defensin 2 (DEFB4, also known as DEFB2 or hBD-2) is a salt-sensitive antimicrobial protein that is expressed in lung epithelia. Previous work has shown that it is encoded in a cluster of beta-defensin genes at 8p23.1, which varies in copy number between 2 and 12 in different individuals. We determined the copy number of this locus in 355 patients with cystic fibrosis (CF), and tested for correlation between beta-defensin cluster genomic copy number and lung disease associated with CF. No significant association was found.

Cystic fibrosis (CF) airway epithelia exhibit raised transepithelial Na+ transport rates, as determined by open-circuit isotope fluxes and estimates of the amiloride-sensitive equivalent short-circuit current (Ieq). To study the contribution of apical and basolateral membrane paths to raised Na+ transport in CF, CF nasal epithelial cultures were studied with double-barreled Na(+)-selective microelectrodes and the Ussing chamber technique. Intracellular Na+ activity (acNa) was 24.1 +/- 1.5 mM (n = 36), a value similar to acNa of normal nasal epithelial cells. Reduction of luminal [Na+] to 3 mM abolished Ieq and reduced acNa. Amiloride (10(-4) M) abolished Ieq but increased acNa from 20 +/- 2 to 36 +/- 7 mM (n = 10). Amiloride-induced increase in acNa was not affected by serosal [Na+] reduction but was blocked by preexposure to reduced luminal [Na+]. Amphotericin B increased Ieq during amiloride exposure, indicating that amiloride did not inhibit NA(+)-K(+)-ATPase. Ouabain abolished Ieq and slowly raised acNa. Reduction of serosal [Na+] led to a decrease in acNa that was blocked by bumetanide. It is concluded that 1) CF airway epithelia exhibit an increased apical membrane Na+ permeability, 2) acNa is regulated to a normal level in CF cells despite increased transcellular Na+ fluxes, 3) the abnormal increase in acNa in response to amiloride is dependent on luminal Na+, 4) Na+ is transported across the basolateral membrane by a bumetanide-sensitive cotransport mechanism, and 5) ouabain inhibits the basolateral Na(+)-K(+)-ATPase, causing slow dissipation of the chemical and electrical gradients across the cell membranes.

BACKGROUND

The goal of this study was to determine the association of multiple antibiotic-resistant Pseudomonas aeruginosa (MARPA) acquisition with lung function decline in patients with cystic fibrosis (CF).

METHODS

Using data from Epidemiologic Study of Cystic Fibrosis (ESCF), we identified patients with spirometry data and MARPA, defined as PA (1) resistant to gentamicin and either tobramycin or amikacin, and (2) resistant to ≥1 antipseudomonal beta lactam. MARPA had to be detected in a respiratory culture after ≥2 years of PA-positive but MARPA-negative respiratory cultures. Multivariable piecewise linear regression was performed to model the annual rate of decline in forced expiratory volume in 1 second (FEV(1)) % predicted 2 calendar years before and after the index year of MARPA detection, adjusting for patient characteristics and CF therapies.

RESULTS

In total, 4349 patients with chronic PA and adequate PFT data were identified; 1111 subsequently developed MARPA, while 3238 patients were PA positive but MARPA negative. Compared with patients who did not acquire MARPA, MARPA-positive patients had lower FEV(1) and received more oral (p<0.013) and inhaled (p<0.001) antibiotic therapy. Mean FEV(1) decline did not change significantly after MARPA detection (-2.22% predicted/year before detection and -2.43 after, p=0.45). There was no relationship between persistent infection or FEV(1) quartile and FEV(1) decline.

CONCLUSIONS

Newly detected MARPA was not associated with a significant change in the rate of FEV(1) decline. These results suggest that MARPA is more likely to be a marker of more severe disease and more intensive therapy, and less likely to be contributing independently to more rapid lung function decline.

We present here a first systematic study of substituent effects in metallocorroles, based on electronic absorption, resonance Raman (RR), and infrared (IR) spectroscopic studies and electrochemical measurements on 10 copper(III) meso-triarylcorroles, Cu(III)[beta-Y(8)TArC], where the beta-substituent Y = H or Br and the meso-aryl group Ar = C(6)F(5) or p-X-C(6)H(4) and X = CF(3), H, CH(3), and OCH(3). The results afford a number of significant inisights. (1) The RR (and IR) results show that at least two and possibly more high-frequency bands in the 1400-1550 cm(-1) region exhibit significant frequency downshifts on beta-octabromination and, thus, qualify as structure-sensitive marker bands. DFT geometry optimizations indicate that the saddled conformation should be clearly preferred for the beta-octabromo-meso-triarylcorrole derivatives studied and that beta-octabromination results in expansion of a number of skeletal bond distances of the corrole macrocycle, consistent with observed frequency downshifts. (2) Electrochemical measurements on planar Cu(III)[TArC] derivatives have shown that the para substituents on the meso-aryl groups exert a strong influence on the half-wave potentials for oxidation (rho(ox) = DeltaE(1/2ox)/Delta(3sigma) = 95 mV), suggesting that oxidation involves removal of an electron from the corrole "b(1)" HOMO, which has significant amplitudes at the meso postions and crudely resembles a porphyrin a(2u) HOMO in shape. In contrast, the Hammett rho(ox) is much lower for the nonplanar Cu(III)[Br(8)TArC] derivatives and we suggest that this ultimately results from a b(1)-to-a(2) HOMO reversal which in turn stems from a metal (d(x2-y2)-corrole ("b(1)") orbital interaction that becomes symmetry-allowed under a saddle distortion of the corrole macrocycle. In contrast to what has been observed for metallotetraphenylporphyrins, beta-octabromination dramatically raises the half-wave potential for one-electron oxidation of the triarylcorrole derivatives studied. This appears to be due to the fact that both the "a(2)" and "b(1)" HOMOs of a corrole (in C(2v) notation) have significantly higher amplitudes at the beta positions, compared to a porphyrin a(2u) HOMO. Thus, although many metallocorroles are significantly more easily oxidizable than analogous metalloporphyrins, certain beta-octahalogeno-meso-triarylcorrole derivatives can indeed be extremely electron deficient and oxidation resistant and may, therefore, find use as rugged catalysts or reagents under highly oxidizing conditions. (3) Finally, the Soret absorption maxima of high-valent metallotriarylcorroles exhibit a uniquely sensitive dependence on the substituents on the meso-aryl groups. Thus, on going from Cu(III)[T(p-CF(3)-P)C] (T(p-CF(3)-P)C = meso-tris((p-trifluoromethyl)phenyl)corrolato) to Cu(III)[T(p-OM-P)C] (T(p-OM-P)C = meso-tris(p-methoxyphenyl)corrolato), the Soret maximum red shifts by 26 nm, from 407 to 433 nm. Similarly, on going from Cu(III)[Br(8)T(p-CF(3)-P)C] (Br(8)T(p-CF(3)-P)C = beta-octabromo-meso-tris((p-trifluoromethyl)phenyl)corrolato) to Cu(III)[Br(8)T(p-OM-P)C] (Br(8)T(p-CF(3)-P)C = beta-octabromo-meso-tris(p-methoxyphenyl)corrolato), the Soret maximum red shifts by 34 nm, from 434 to 468 nm. Time-dependent DFT calculations suggest that this substituent dependence reflects significant ligand-to-metal charge-transfer character of certain transitions in the Soret region. The optical spectra of free-base and non-high-valent transition metal tetrapyrroles, in general, do not exhibit a similar substituent dependence.

Generation and precise genetic correction of patient-derived hiPSCs have great potential in regenerative medicine. Such targeted genetic manipulations can now be achieved using gene-editing nucleases. Here, we report generation of cystic fibrosis (CF) and Gaucher's disease (GD) hiPSCs respectively from CF (homozygous for CFTRΔF508 mutation) and Type II GD [homozygous for β-glucocerebrosidase (GBA) 1448T>C mutation] patient fibroblasts, using CCR5- specific TALENs. Site-specific addition of loxP-flanked Oct4/Sox2/Klf4/Lin28/Nanog/eGFP gene cassette at the endogenous CCR5 site of patient-derived disease-specific primary fibroblasts induced reprogramming, giving rise to both monoallele (heterozygous) and biallele CCR5-modified hiPSCs. Subsequent excision of the donor cassette was done by treating CCR5-modified CF and GD hiPSCs with Cre. We also demonstrate site-specific correction of sickle cell disease (SCD) mutations at the endogenous HBB locus of patient-specific hiPSCs [TNC1 line that is homozygous for mutated β- globin alleles (βS/βS)], using HBB-specific TALENs. SCD-corrected hiPSC lines showed gene conversion of the mutated βS to the wild-type βA in one of the HBB alleles, while the other allele remained a mutant phenotype. After excision of the loxP-flanked DNA cassette from the SCD-corrected hiPSC lines using Cre, we obtained secondary heterozygous βS/βA hiPSCs, which express the wild-type (βA) transcript to 30-40% level as compared to uncorrected (βS/βS) SCD hiPSCs when differentiated into erythroid cells. Furthermore, we also show that TALEN-mediated generation and genetic correction of disease-specific hiPSCs did not induce any off-target mutations at closely related sites.

Cardiac fibrosis is a major cause of heart failure. MicroRNAs (miRs) are important epigenetic regulators of cardiac function and cardiovascular diseases, including cardiac fibrosis. This study aimed to explore the role of miR-503 and its mechanisms in regulating cardiac fibrosis. miR-503 was found up-regulated in the mouse LV tissues subjected to transverse aortic constriction (TAC) and in neonatal cardiac fibroblasts (CFs) cultured with Angiotension II. The role of miR-503 in regulating CF cell proliferation and/or collagen production in mice neonatal CFs were determined using an MTT assay and RT-PCR respectively. Forced expression of miR-503 increased the cellular proliferation and collagen production in mice neonatal CFs. The effects were abrogated by cotransfection with AMO-503 (a specific inhibitor of miR-503). Injection of antagomiR-503 elevated cardiac function and inhibited the expression of connective tissue growth factor (CTGF) and transforming growth factor (TGF)-β in the TAC mice. Additional analysis revealed that Apelin-13 is a direct target of miR-503, as the overexpression of miR-503 decreased the protein and mRNA expression levels of Apelin-13. In the CFs with pre-treatment of AngII, we transfected AMO-503 into the cells treated with siRNA-APLN. siRNA-APLN abolished the effects of AMO-503 on the production of collagen I and III and the expression of TGF-β and CTGF. Furthermore, pre-treatment of CFs with Apelin-13 (1-100 nmol/l) inhibited angiotensin II-mediated collagen production and activation of CTGF and TGF-β. So we conclude that miR-503 promotes cardiac fibrosis via miR-503-Apelin-13-TGF-β-CTGF-collagen production pathway. Thus, miR-503 is a promising therapeutic target for reducing cardiac fibrosis.

There is increasing evidence that reactive oxygen species (ROS) contribute to post-ischemic reperfusion injury, but determination of the specific ROS involved has proven elusive. In the present study electron paramagnetic resonance (EPR) spectroscopy was used in vitro to measure the relative quenching of singlet oxygen (1O2) by histidine and carnosine (beta-alanyl-L-histidine) utilizing the hindered secondary amine 2,2,6,6-tetramethyl-4-piperidone HCl (4-oxo-TEMP). The relative effect of histidine and carnosine on functional recovery of isolated perfused rat hearts was also studied. Functional recovery was measured by left ventricular developed pressure (LVDP), first derivative of left ventricular pressure (dP/dt), heart rate (HR) and coronary flow (CF). EPR measurements and Stern-Volmer plots showed that 400 microM carnosine quenched 1O2 twice as effectively as equimolar histidine in vitro. Moreover, 10 mM histidine improved functional recovery of isolated rat hearts significantly more than 1 mM histidine. Furthermore, 1 mM carnosine improved functional recovery significantly more than equimolar histidine and as effectively as 10 mM histidine. Experiments with 1 mM mannitol, a known hydroxyl radical scavenger, did not show an improvement in functional recovery relative to control hearts, thereby decreasing the likelihood that hydroxyl radicals are the major damaging species. On the other hand, the correlation between improved functional recovery of isolated rat hearts with histidine and carnosine and their relative 1O2 quenching effectiveness in vitro provides indirect evidence for 1O2 as ROS participating in reperfusion injury.

Pneumothorax is recognized as a common and life-threatening complication in cystic fibrosis (CF) patients, especially in those who are infected with P. aeruginosa, B. cepacia or Aspergillus, need enteral feeding, are diagnosed as suffering from allergic bronchopulmonary aspergillosis (ABPA), developed massive hemoptysis, and their respiratory function is seriously compromised. Structural impairment and altered airflow dynamics in the lungs of CF patients are considered as the main predisposing factors, but also inhaled medications and non-invasive positive pressure ventilation (NIPPV) could increase the risk of pneumothorax. Clinical presentation could range from dramatic to very mild. Management of spontaneous pneumothorax occurring to patients with CF is essentially similar to that for non-CF patients. Therapeutic options include intercostal tube drainage, video-assisted thoracoscopic surgery (VATS), and medical or surgical pleurodesis. Pneumothorax increases both short- and long-term morbidity and mortality in CF patients and causes significant deterioration of their quality of life.

Both the enzymes of peroxisomal fatty acid beta-oxidation and the liver fatty acid-binding protein (L-FABP) are induced in the liver by peroxisome proliferators, such as clofibrate (CF), as well as high fat diets. One proposed mechanism for this induction is that it represents an adaptive response to altered intracellular fatty acid fluxes, mediated by dicarboxylic fatty acids formed via the cytochrome P-450IVA1 omega-oxidation pathway. The studies presented in this paper were designed to investigate the role of the products of P-450IVA1 omega-oxidation in the regulation of peroxisomal beta-oxidation and L-FABP. In primary hepatocyte cultures exposed to CF, the increase in P-450IVA1 activity preceded the induction of peroxisomal beta-oxidation and L-FABP. The CF-mediated increases in peroxisomal beta-oxidation and L-FABP, but not P-450IVA1, could be significantly inhibited pretranslationally by concurrent exposure of cultured hepatocytes to inactivators of cytochromes P-450, such as 1-aminobenzotriazole and 10-undecynoic acid. Hexadecanedioic acid, a 16-carbon dicarboxylic fatty acid, that is poorly metabolized in hepatocytes, induced peroxisomal beta-oxidation and L-FABP, but not P-450IVA1, via a pretranslational mechanism that was not inhibited by 1-aminobenzotriazole. Long-chain monocarboxylic acids were without such inducing effect. In further studies, non-beta-oxidizable dicarboxylic acid analogs were found to display greater potency as inducers of peroxisomal beta-oxidation when compared to hexadecanedioic acid. The inducing effects of the dicarboxylic acid analogs were also independent of the P-450 omega-oxidation pathway. The results of these studies suggest that the regulation of peroxisomal beta-oxidation enzymes and L-FABP is mediated, to a significant extent, by poorly metabolized long-chain dicarboxylic acids formed via the P-450IVA1 pathway.