Recent studies have shown that airway inflammation dominated by neutrophils, ie, polymorphonuclear cells (PMN) was observed in infants and children with cystic fibrosis (CF) even in the absence of detectable infection. To assess whether there is a CF-related anomaly of PMN migration across airway epithelial cells, we developed an in vitro model of chemotactic migration across tight and polarized CF(15) cells, a CF human nasal epithelial cell line, seeded on porous filters. To compare PMN migration across a pair of CF and control monolayers in the physiological direction, inverted CF(15) cells were infected with increasing concentrations of recombinant adenoviruses containing either the normal cystic fibrosis transmembrane conductance regulator (CFTR) cDNA, the DeltaF508 CFTR cDNA, or the beta-galactosidase gene. The number of PMN migrating in response to N-formyl-Met-Leu-Phe across inverted CF(15) monolayers expressing beta-galactosidase was similar to that seen across CF(15) monolayers rescued with CFTR, whatever the proportion of cells expressing the transgene. Moreover, PMN migration across monolayers expressing various amounts of mutated CFTR was not different from that observed across matched counterparts expressing normal CFTR. Finally, PMN migration in response to adherent or Pseudomonas aeruginosa was equivalent across CF and corrected monolayers. The possibility that mutated CFTR may exert indirect effects on PMN recruitment, via an abnormal production of the chemotactic cytokine interleukin-8, was also explored. Apical and basolateral production of interleukin-8 by polarized CF cells expressing mutated CFTR was not different from that observed with rescued cells, either in baseline or stimulated conditions. CF(15) cells displayed a CF phenotype that could be corrected by CFTR-containing adenoviruses, because two known CF defects, Cl(-) secretion and increased P. aeruginosa adherence, were normalized after infection with those viruses. Thus, we conclude that the presence of a mutated CFTR does not per se lead to an exaggerated inflammatory response of CF surface epithelial cells in the absence or presence of a bacterial infection.

(<em>b</em>)Purpose:</<em>b</em>) Both cardiomyocytes and cardiac fi<em>b</em>ro<em>b</em>lasts (<em>CF</em>) play essential roles in cardiac development, function, and remodeling. Properties of 3D co-cultures are incompletely understood. Hence, 3D co-culture of cardiomyocytes and <em>CF</em> was characterized, and selected features compared with single-type and 2D culture conditions. (<em>b</em>)Methods:</<em>b</em>) Human cardiomyocytes derived from induced-pluripotent stem cells (hiPSC-CMs) were o<em>b</em>tained from Cellular Dynamics or Ncardia, and primary human cardiac fi<em>b</em>ro<em>b</em>lasts from ScienCell. Cardiac spheroids were investigated using cryosections and whole-mount confocal microscopy, video motion analysis, scanning-, and transmission-electron microscopy (SEM, TEM), action potential recording, and quantitative PCR (qPCR). (<em>b</em>)Results:</<em>b</em>) Spheroids formed in hanging drops or in non-adhesive wells showed spontaneous contractions for at least 1 month with frequent media changes. SEM of mechanically opened spheroids revealed a dense inner structure and no signs of <em>b</em>le<em>b</em><em>b</em>ing. TEM of co-culture spheroids at 1 month showed myofi<em>b</em>rils, intercalated disc-like structures and mitochondria. Ultrastructural features were compara<em>b</em>le to fetal human myocardium. We then assessed immunostained 2D cultures, cryosections of spheroids, and whole-mount preparations <em>b</em>y confocal microscopy. <em>CF</em> in co-culture spheroids assumed a small size and shape similar to the situation in ventricular tissue. Spheroids made only of <em>CF</em> and cultured for 3 weeks showed no stress fi<em>b</em>ers and strongly reduced amounts of alpha smooth muscle actin compared to early spheroids and 2D cultures as shown <em>b</em>y confocal microscopy, western <em>b</em>lotting, and qPCR. The addition of <em>CF</em> to cardiac spheroids did not lead to arrhythmogenic effects as measured <em>b</em>y sharp-electrode electrophysiology. Video motion analysis showed a faster spontaneous contraction rate in co-culture spheroids compared to pure hiPSC-CMs, <em>b</em>ut similar contraction amplitudes and kinetics. Spontaneous contraction rates were not dependent on spheroid size. Applying increasing pacing frequencies resulted in decreasing contraction amplitudes without positive staircase effect. Gene expression analysis of selected cytoskeleton and myofi<em>b</em>rillar proteins showed more tissue-like expression patterns in co-culture spheroids than with cardiomyocytes alone or in 2D culture. (<em>b</em>)Conclusion:</<em>b</em>) We demonstrate that the use of 3D co-culture of hiPSC-CMs and <em>CF</em> is superior over 2D culture conditions for co-culture models and more closely mimicking the native state of the myocardium with relevance to drug development as well as for personalized medicine.

CONCLUSIONS

Irreversible tissue damage within the cystic fibrosis (CF) lung is mediated by proteolytic enzymes during an inflammatory response. Serine proteinases, in particular neutrophil elastase (NE), have been implicated however, members of the cysteine proteinase family may also be involved. The aim of this study was to determine cathepsin B and S levels in cystic fibrosis (CF) sputum and to assess any relationship to recognized markers of inflammation such as sputum NE, interleukin-8 (IL-8), tumor necrosis factor alpha (TNF-alpha), urine TNF receptor 1 (TNFr1), plasma IL-6, and serum C-reactive protein (CRP). Proteinase activities were measured in the sputum of 36 clinically stable CF patients using spectrophotometric and fluorogenic assays. Immunoblots were also used to confirm enzyme activity data. All other parameters were measured by ELISA. Patients had a mean age of 27.2 (8.2) years, FEV. of 1.6 (0.79) L and BMI of 20.7 (2.8). Both cathepsin B and S activities were detected in all samples, with mean concentrations of 18.0 (13.5) microg/ml and 1.6 (0.88) microg/ml, respectively and were found to correlate not only with each other but with NE, TNF-alpha and IL-8 (in all cases . < 0.05). Airway cathepsin B further correlated with circulatory IL-6 and CRP however, no relationship for either cathepsin was observed with urine TNFr1. This data indicates that cathepsin B and S may have important roles in the pathophysiology of CF lung disease and could have potential as markers of inflammation in future studies.

Three experiments investigated whether and to what extent increases in age affect the functionality of stereopsis. The observers' ages ranged from 18 to 83 years. The overall goal was to challenge the older stereoscopic visual system by utilizing high magnitudes of binocular disparity, ambiguous binocular disparity [cf., Julesz, B., & Chang, J. (1976). Interaction between pools of binocular disparity detectors tuned to different disparities. Biological Cybernetics, 22, 107-119], and by making binocular matching more difficult. In particular, Experiment 1 evaluated observers' abilities to discriminate ordinal depth differences away from the horopter using standing disparities of 6.5-46 min arc. Experiment 2 assessed observers' abilities to discriminate stereoscopic shape using line-element stereograms. The direction (crossed vs. uncrossed) and magnitude of the binocular disparity (13.7 and 51.5 min arc) were manipulated. Binocular matching was made more difficult by varying the orientations of corresponding line elements across the two eyes' views. The purpose of Experiment 3 was to determine whether the aging stereoscopic system can resolve ambiguous binocular disparities in a manner similar to that of younger observers. The results of all experiments demonstrated that older observers' stereoscopic vision is functionally comparable to that of younger observers in many respects. For example, both age groups exhibited a similar ability to discriminate depth and surface shape. The results also showed, however, that age-related differences in stereopsis do exist, and they become most noticeable when the older stereoscopic system is challenged by multiple simultaneous factors.

M. dilatata, M. flabellata, and M. patula and 80 other scleractinian corals were petitioned to be listed under the US Endangered Species Act (ESA), which would have major conservation implications. One of the difficulties with this evaluation is that reproductive boundaries between morphologically defined coral species are often permeable, and morphology can be wildly variable. We examined genetic and morphological variation in Hawaiian Montipora with a suite of molecular markers (mitochondrial: COI, CR, Cyt-B, 16S, ATP6; nuclear: ATPsβ, ITS) and microscopic skeletal measurements. Mitochondrial markers and the ITS region revealed four distinct clades: I) M. patula/M. verrilli, II) M. cf. incrassata, III) M. capitata, IV) M. dilatata/M. flabellata/M. cf. turgescens. These clades are likely to occur outside of Hawai'i according to mitochondrial control region haplotypes from previous studies. The ATPsβ intron data showed a pattern often interpreted as resulting from hybridization and introgression; however, incomplete lineage sorting may be more likely since the multicopy nuclear ITS region was consistent with the mitochondrial data. Furthermore, principal components analysis (PCA) of skeletal microstructure was concordant with the mitochondrial clades, while nominal taxa overlapped. The size and shape of verrucae or papillae contributed most to identifying groups, while colony-level morphology was highly variable. It is not yet clear if these species complexes represent population-level variation or incipient speciation (CA<1MYA), two alternatives that have very different conservation implications. This study highlights the difficulty in understanding the scale of genetic and morphological variation that corresponds to species as opposed to population-level variation, information that is essential for conservation and for understanding coral biodiversity.

Circulating antibodies can be used to probe protein arrays of body fluids, prepared by two-dimensional gel electrophoresis, for antigenic biomarker detection. However, detected proteins, particularly low abundance antigens, often remain unidentifiable due to proteome complexity and limiting sample amounts. Using a novel enrichment approach exploiting patient antibodies for isolation of antigenic biomarkers, we demonstrate how immunoproteomic strategies can accelerate biomarker discovery. Application of this approach as a means of identifying biomarkers was demonstrated for cystic fibrosis (CF) lung disease by isolation and identification of inflammatory-associated autoantigens, including myeloperoxidase and calgranulin B from sputum of subjects with CF. The approach was also exploited for isolation of proteins expressed by the Pseudomonas aeruginosa strain PA01. Capture of PA01 antigens using circulating antibodies from CF subjects implicated in vivo expression of Pseudomonas proteins. All CF subjects screened, but not controls, were immunoreactive against immunocaptured Pseudomonas proteins, representing stress (GroES and ferric iron-binding protein HitA), immunosuppressive (thioredoxin), and alginate synthetase pathway (nucleoside-diphosphate kinase) proteins, implicating their clinical relevance as biomarkers of infection.

Exosomes derived from human umbilical cord mesenchymal stem cells (hucMSCs) are a promising new therapeutic option for myocardial infarction (MI). The tissue matrix metalloproteinase inhibitor 2, also known as TIMP2, is a member of the tissue inhibitor family of metalloproteinases. Since TIMP2-mediated inhibition of matrix metalloproteinases (MMPs) is a key determinant of post-MI remodeling, we analyzed the therapeutic effects of exosomes derived from TIMP2-overexpressing hucMSCs (huc-exo^TIMP2) on the MI rat model. The huc-exo^TIMP2 significantly improved in vivo cardiac function as measured by echocardiography and promoted angiogenesis in MI injury. It also restricted extracellular matrix (ECM) remodeling, as indicated by the reduced collagen deposition. In addition, huc-exo^TIMP2 administration increased the in situ expression of the antiapoptotic Bcl-2 and decreased that of the proapoptotic Bax and pro-caspase-9 in the infracted myocardium. Meanwhile, huc-exo^TIMP2 upregulated superoxide dismutase (SOD) as well as glutathione (GSH) and decreased the malondialdehyde (MDA) level in MI models. In vitro huc-exo^TIMP2 pretreatment could inhibit H₂O₂-mediated H9C2-cardiomyocyte apoptosis and promote human umbilical vein endothelial cell (HUVEC) proliferation, migration, and tube formation, as well as decrease TGFβ-induced MMP2, MMP9, and α-SMA secretion by cardiac fibroblasts (CFs). Besides that, huc-exo^TIMP2 pretreatment also increased the expression of Akt phosphorylation in the infarcted myocardium, which may relate to a high level of secreted frizzled-related protein 2 (Sfrp2) in huc-exo^TIMP2, indicating a mechanistic basis of its action. Importantly, Sfrp2 knockdown in huc-exo^TIMP2 abrogated the protective effects. Taken together, huc-exo^TIMP2 improved cardiac function by alleviating MI-induced oxidative stress and ECM remodeling, partly via the Akt/Sfrp2 pathway.

OBJECTIVE

To examine oxidative stress in CF by measuring 8-iso-PGF2alpha and antioxidant defenses, in relation to dietary intake, immune function and clinical status.

METHODS

We measured total plasma concentrations of 8-iso-PGF2alpha and dietary antioxidants (vitamin E, vitamin C, beta-carotene), erythrocyte antioxidant enzyme activities (glutathione peroxidase and superoxide dismutase), lung function and dietary intake in 21 CF subjects and 21 healthy age- and gender-matched controls.

RESULTS

Total plasma 8-iso-PGF2alpha concentration (median [quartile 1-quartile 3]) was significantly higher in CF subjects compared to controls (214 pg/mL (155-331) vs. 135 pg/mL (101-168), p = 0.001). Neutrophil, monocyte and total white cell counts were elevated in the CF group and these correlated with 8-iso-PGF2alpha concentration. Despite similar dietary intake, lower plasma antioxidant concentrations were observed in the CF group (vitamin E, p < 0.001, vitamin C, p = 0.004, beta-carotene, p = 0.001). 8-iso-PGF2alpha correlated negatively with plasma vitamin E, C and beta-carotene concentrations.

CONCLUSIONS

Oxidative stress is increased in CF patients, despite normal dietary antioxidant intake. The immune response appears to be a key factor causing oxidative stress. Antioxidant intervention aimed at reducing oxidative stress in CF needs to be assessed.

Inhaled fluticasone propionate (FP) is widely used to reduce pulmonary inflammation in chronic obstructive pulmonary disease, but the potential effects of FP on airway epithelial cells from patients with cystic fibrosis (CF) are unknown. In CF disease, a nonregulated inflammatory lung response occurs through exaggerated nuclear factor (NF)-kappaB activation and elevated pro-inflammatory cytokines production by airway epithelial cells. To determine whether FP reduces cytokine production in bronchial epithelial cells via NF-kappaB, the authors investigated the nonstimulated and the Pseudomonas aeruginosa lipopolysaccharide (LPS) stimulated production of NF-kappaB-dependent interleukin (IL)-6, IL-8 and RANTES (regulated on activation, T-cell expressed and secreted) along with the activation of NF-kappaB in non-CF and CF human bronchial gland epithelial cells. It was demonstrated that a relevant concentration of FP (10(-8) M) inhibited constitutive and P. aeruginosa LPS-induced IL-6 and IL-8 production of non-CF and CF bronchial epithelial cells. Interestingly, the expression of two IkappaB kinases (IKK)-alpha/beta, the degradation of cytosolic IkappaB-beta inhibitor and the NF-kappaB deoxyribonucleic acid binding activity were markedly reduced after FP treatment in both CF and non-CF bronchial epithelial cells. It was shown by the authors that fluticasone propionate exerts an anti-inflammatory effect by blocking a signal transduction leading to a reduced level of IkappaB-alpha/beta kinases in bronchial epithelial cells. In particular the strong effect on the IkappaB-beta kinase, which is known to be elevated in bronchial epithelial cells in cystic fibrosis patients, was observed.

The aim of this study was to investigate a possible reenhancement of islet cell autoimmunity in type I (insulin-dependent) diabetic patients who received HLA-mismatched pancreas transplants from cadaveric donors and who underwent generalized immunosuppression. Circulating islet cell antibodies (ICA) and complement-fixing ICAs (CF-ICAs) have been tested at 1, 2, 3, 6, and 12 mo and at least once a year posttransplantation in 23 recipients of 25 transplants (22 simultaneous with kidney, 2 retransplants, 1 isolated; 23 segmental neoprene injected, 2 whole with enteric drainage). Patients were aged 35.3 +/- 1.9 yr with a duration of diabetes of 20.6 +/- 1.1 yr. Immunosuppression consisted of double or triple association of azathioprine, cyclosporin, and prednisone with or without temporary antilymphocyte globulins. The number of HLA-A and HLA-B compatibilities was none in 8 patients, one in 12 patients, two in 4 patients, and three in 1 patient. The mean follow-up was 4.0 +/- 0.4 yr/patient (range 0.4-7.2). ICAs were positive pretransplantation in 2 of 25 patients and reappeared 1-42 mo posttransplantation in another 7. In 6 patients, CF-ICAs were also positive. In 7 of 9 ICA+ patients the pancreas transplant failed; in 1 patient this occurred 4 mo before ICA reappearance, and in 6 patients it occurred 2-35 mo after the first detection of ICAs. Pancreas-transplant failure was significantly associated with the positivity for ICAs (P less than .05) and particularly for CF-ICAs (P less than .005). ICA positivity was transitory in 4 patients (2-27 mo) and persistent in the remaining 5 (up to 61 mo).(ABSTRACT TRUNCATED AT 250 WORDS)

Citrullinated proteins, derived from the conversion of peptidyl-arginine to peptidyl-citrulline, are present in the joints of patients with rheumatoid arthritis (RA), who also uniquely produce high levels of anti-citrullinated protein Abs. Citrullinated fibrinogen (CF) is abundant in rheumatoid synovial tissue, and anti-citrullinated protein Ab-positive RA patients exhibit circulating immune complexes containing CF. Thus, CF is a potential major target of pathogenic autoimmunity in RA. T cells are believed to be involved in this process by initiating, controlling, and driving Ag-specific immune responses in RA. In this study, we isolated a CD4 T cell line specific for CF that produces inflammatory cytokines. When transferred into mice with collagen-induced arthritis (CIA), this T cell line specifically enhanced the severity of autoimmune arthritis. Additionally, pathogenic IgG2a autoantibody levels to mouse type II collagen were increased in mice that received the T cells in CIA, and levels of these T cells were increased in the synovium, suggesting the T cells may have had systemic effects on the B cell response as well as local effects on the inflammatory environment. This work demonstrates that CD4 T cells specific for CF can amplify disease severity after onset of CIA.

OBJECTIVE

In order to gain a better understanding of the role of several mechanisms in antibiotic resistance in Pseudomonas aeruginosa clinical isolates obtained from CF and burn patients, we evaluated gene expression of efflux pumps MexAB-OprM and MexXY(-OprA), the natural β-lactamase AmpC and outer membrane porin protein OprD. Also, the presence of genes encoding Ambler classes A, B β-lactamases and aminoglycoside modifying enzymes (AMEs) was examined.

RESULTS

Piperacillin-tazobactam and amikacin retained the highest in vitro activities among 21 CF and 27 burn P. aeruginosa isolates. Based on Enterobacterial Repetitive Intergenic Consensus (ERIC) PCR, 15 distinct patterns were detected. There were 5 CF and 6 burn isolates harbored PER-1 and VEB-1, respectively. Among AMEs, involved in resistance of anti-Pseudomonas aminoglycosides, aac(6')-Ib was the most prevalent gene. Among CF isolates, mexA overexpression was the most prevalent mechanism (47.6%) followed by mexX (42.8%), ampC (9.5%) and oprD downregulation (4.7%). Among burn isolates, the prevalence of mexX, mexA, and ampC overexpression was 62.9%, 74%, and 11.1%, respectively. Downregulation of oprD was observed in 14.8% of burn isolates.

CONCLUSIONS

Among CF isolates, mexX and mexA overexpression were the major contributing factors to aminoglycoside (gentamicin) and carbapenem (meropenem) resistance, respectively while among burn isolates, AMEs in conjunction with mexX hyperexpression were identified to be responsible for aminoglycoside resistance. Also mexA overexpression was partially associated with carbapenem resistance. Moreover, cephalosporin resistance was linked to overexpression of mexA and/or mexX. The impact of interplay between different resistance mechanisms on resistant phenotypes was more complicated among burn than CF isolates.

Chemical analysis of a specimen of the sponge Ianthella cf. flabelliformis returned two new sesquiterpene glycinyl lactams, ianthellalactams A (1) and B (2), the known sponge sesquiterpene dictyodendrillin (3) and its ethanolysis artifact ethyl dictyodendrillin (4), and five known sponge indole alkaloids, aplysinopsin (5), 8E-3'-deimino-3'-oxoaplysinopsin (6), 8Z-3'-deimino-3'-oxoaplysinopsin (7), dihydroaplysinopsin (8) and tubastrindole B (9). The equilibrated mixture 6/7 exhibited glycine-gated chloride channel receptor (GlyR) antagonist activity with a bias towards α3 over α1 GlyR, while tubastrindole B (9) exhibited a bias towards α1 over α3 GlyR. At low- to sub-micromolar concentrations, 9 was also a selective potentiator of α1 GlyR, with no effect on α3 GlyR-a pharmacology that could prove useful in the treatment of movement disorders such as spasticity and hyperekplexia. Our investigations into the GlyR modulatory properties of 1-9 were further supported by the synthesis of a number of structurally related indole alkaloids.

BACKGROUND

Chronic airway infection by Pseudomonas aeruginosa considerably contributes to lung tissue destruction and impairment of pulmonary function in cystic-fibrosis (CF) patients. Complex interplays between P. aeruginosa and other co-colonizing pathogens including Staphylococcus aureus, Burkholderia sp., and Klebsiella pneumoniae may be crucial for pathogenesis and disease progression.

METHODS

We generated a library of PA14 transposon insertion mutants to identify P. aeruginosa genes required for exploitative and direct competitions with S. aureus, Burkholderia cenocepacia, and K. pneumoniae.

RESULTS

Whereas wild-type PA14 inhibited S. aureus growth, two transposon insertions located in pqsC and carB, resulted in reduced growth inhibition. PqsC is involved in the synthesis of 4-hydroxy-2-alkylquinolines (HAQs), a family of molecules having antibacterial properties, while carB is a key gene in pyrimidine biosynthesis. The carB mutant was also unable to grow in the presence of B. cepacia and K. pneumoniae but not Escherichia coli and S. epidermidis. We further identified a transposon insertion in purF, encoding a key enzyme of purine metabolism. This mutant displayed a severe growth deficiency in the presence of Gram-negative but not of Gram-positive bacteria. We identified a beneficial interaction in a bioA transposon mutant, unable to grow on rich medium. This growth defect could be restored either by addition of biotin or by co-culturing the mutant in the presence of K. pneumoniae or E. coli.

CONCLUSIONS

Complex interactions take place between the various bacterial species colonizing CF-lungs. This work identified both detrimental and beneficial interactions occurring between P. aeruginosa and three other respiratory pathogens involving several major metabolic pathways. Manipulating these pathways could be used to interfere with bacterial interactions and influence the colonization by respiratory pathogens.

The Burkholderia cepacia complex (Bcc) is a heterogeneous group of bacteria comprising around 20 related species. These bacteria are important opportunistic pathogens, especially in cystic fibrosis (CF) patients, and are associated with a worse prognosis and decreased life expectancy. The taxonomic position of 20 Bcc isolates retrieved from CF patients receiving care at Hospital Santa Maria (HSM), in Lisbon, from 1995 to 2006, was re-examined in the present work. These isolates, formerly classified as Burkholderia cepacia (taxon K), are here reclassified as Burkholderia contaminans, including the former B. cepacia IST408, which was the focus of previous studies regarding the biosynthesis of the exopolysaccharide 'cepacian'. The CF population examined has been previously described as having an exceptionally high representation of B. cepacia, presumably due to a contamination arising from saline solutions for nasal application. Twenty-one additional isolates, obtained from a chronically infected patient, from 2006 to 2010, were also identified as B. contaminans. This study also provides insight into the potential clinical impact of B. contaminans, a species that is rarely associated with CF infections. Isolates belonging to this species were shown to be involved in chronic and transient respiratory infections, and were associated with severe lung function deterioration and with a case of death with cepacia syndrome. However, since the patients were co-infected with Burkholderia cenocepacia and other non-Burkholderia bacteria, the role played by B. contaminans is unclear. Nevertheless, B. contaminans isolates were found to prevail over B. cenocepacia isolates during co-infection of at least one chronically infected patient.

Since conducting follow-up studies of patients with acute symptomatic parvovirus <em>B</em>19 infection which showed that a significant proportion of patients develop prolonged arthritis and chronic fatigue syndrome (<em>CFS</em>), we have become interested in the mechanisms of this phenomenon. We showed that these cases have high levels of pro-inflammatory cytokines in their circulation and that this correlates with the symptoms. However, the underlying mechanisms were not apparent, and we have used various approaches to begin studying this phenomenon. DNA polymorphisms were looked for and several were shown to be more common in these subjects compared with controls; these occur within genes of both the immune response [human leucocyte antigen (HLA)-DR<em>B</em>1, HLA-<em>B</em>, transforming growth factor (TGF)-beta1] and those involved in several other cellular functions (predominantly the cytoskeleton and cell adhesion). Interestingly, one particular single-nucleotide polymorphism (SNP) which is associated with symptomatic <em>B</em>19 infection occurs in the Ku80 gene which has recently been shown to be a <em>B</em>19 co-receptor. <em>B</em>19 persistence is probably the key to this phenomenon, and some new data are presented on short regions of sequence homology (17-26 bp) between human, mouse and rat parvoviruses and their respective hosts which occur in many host genes. This homology may provide a foothold for virus persistence and may also play a role in the genesis of disease through gene disruption. Finally, we used microarrays and TaqMan real-time polymerase chain reaction in 108 normal persons to study human gene expression in persons who are <em>B</em>19-seropositive versus <em>B</em>19-seronegative (age- and sex-matched) to examine the hypothesis that gene regulation may be altered in subjects harbouring the <em>B</em>19 virus DNA. Six genes were found to be differentially expressed with roles in the cytoskeleton (SKIP, MACF1, SPAG7, FLOT1), integrin signalling (FLOT1, RASSF5), HLA class III (c6orf48), and tumour suppression (RASSF5). These results have implications not only for <em>B</em>19 but also for other persistent viruses as well and confirmation is required. In conclusion, these disparate findings contribute to our understanding of the pathogenesis of <em>B</em>19 disease. We are using these studies as a starting point to study the phenomenon of chronic immune activation following <em>B</em>19 infection.

Relapse after apparently successful treatment of coccidioidomycosis has been a problem with both amphotericin B and the azoles. We conducted a retrospective cohort study of 34 patients who required therapy for coccidioidomycosis between 1973 and 1993; 10 relapsed and 25 (one patient received two courses of therapy) did not relapse during follow-up. The mean time to relapse after completion of therapy was 7.3 months (range, 1-21 months). All 34 patients responded clinically to therapy. A fourfold or greater decrease in titers of antibody, as determined by complement fixation (CF), during therapy was seen in seven (78%) of nine patients who relapsed and 17 (85%) of 20 patients who did not relapse (P = .956). There was no significant difference between relapsers and nonrelapsers in terms of the lowest CF titer during therapy, the CF titer at the end of therapy, or the peak CF titer. The risk of relapse was increased among those with a peak CF titer of>> or = 1:256 (relative risk [RR] = 4.7; 95% confidence interval [CI] = 1.4-16.1), as compared with patients who did not mount such a high antibody response. Similarly, the risk of relapse was higher among those with serially negative coccidioidin skin tests (CSTs) than those with serially positive CSTs (RR = 4.8; 95% CI = 1.2-19.5). We conclude that clinical response, lowest CF titer, end-of-therapy CF titer, and decrease in the CF titer of at least fourfold are not predictive of relapse in patients with coccidioidomycosis. Negative serial coccidioidin skin tests and a peak CF antibody titer of>> or = 1:256 are independently associated with increased risk of relapse.

BACKGROUND

Continuous flow (CF) left ventricular assist devices (LVAD) are afterload sensitive and therefore pump performance is affected by hypertension. In addition, poorly controlled hypertension may increase the risk of aortic insufficiency (AI) and stroke. Blood pressure regimens after CF LVAD have not been studied and their impact on rates of AI and stroke are unknown.

METHODS

Patients who had CF LVAD at a single center and were supported greater than 30 days were included. Blood pressure was monitored at home by Doppler. Outpatient management of blood pressure was conducted according to a predefined institutional protocol (target mean arterial pressure ≤ 80 mm Hg).

RESULTS

A total of 96 patients were included. At the end of follow-up, 25 patients were not on an antihypertensive drug, of these 9 died. Of the 74% receiving antihypertensives, 54% required 1 medication, 34% were on 2, 10% were on 3, and 3% were on 4 or more. Angiotensin-converting enzyme inhibitors or angiotensin II receptor blockers (85% of patients on an antihypertensive) and beta blockers (30%) were the most commonly prescribed medications. There was a significantly higher neurologic event rate in those on no antihypertensives compared with those on antihypertensives (p = 0.009). Only 3% of patients with no or mild AI at baseline progressed to develop moderate or greater AI after a mean of 201 days of follow-up.

CONCLUSIONS

Blood pressure control can be achieved in patients with CF LVADs, with the majority of patients requiring only 1 or 2 antihypertensives.

We studied changes in the epilimnetic bacterial community composition (BCC), bacterial biomass and production, and protistan succession and bacterivory along the longitudinal axis of the canyon-shaped, highly eutrophic Sau Reservoir (NE Spain) during two sampling campaigns, in April and July 1997. Longitudinal changes in BCC from the river inflow to the dam area of the reservoir were detected by using oligonucleotide probes targeted to the kingdom Bacteria, to the alpha, beta, and gamma subclasses (ALFA, BETA, and GAMA) of the class Proteobacteria, and to the Cytophaga/Flavobacterium (CF) cluster. In general, the inflow of the organically loaded Ter river, with highly abundant allochthonous bacterial populations, induced a clearly distinguishable longitudinal succession of the structure of the microbial food web. The most dynamic changes in microbial parameters occurred at the plunge point, the mixing area of river water and the reservoir epilimnion. Changes within members of BETA and CF were the most important in determining changes in BCC, bacterial abundance and biomass. Much less relevant changes occurred within the less abundant ALFA and GAMA bacteria. From the plunge point downstream, we described a significant shift in BCC in the form of decreased proportions of BETA and CF. This shift spatially coincided with the highest values of heterotrophic nanoflagellate bacterivory (roughly doubled the bacterial production). CF numerically dominated throughout the reservoir without any marked longitudinal changes in their mean cell volume. In contrast, very large cells affiliated to BETA clearly dominated in the allochthonous bacterial biomass brought by the river. BETA showed a marked downstream trend of decreasing mean cell volume. We conclude that the observed BCC shift and the longitudinal shift in food web structure (bacteria-heterotrophic nanoflagellates-ciliates) resulted from highly complex interactions brought about by several major factors: varying hydrology, the high localized allochthonous input of organic matter brought by the river, downstream changing substrate availability, and selective protistan bacterivory.

Tracheal glands (TG) may play a specific role in the pathogenesis of cystic fibrosis (CF), a disease due to mutations in the cftr gene and characterized by airway inflammation and Pseudomonas aeruginosa infection. We compared the gene expression of wild-type TG cells and TG cells with the cftr DeltaF508 mutation (CF-TG cells) using microarrays covering the whole human genome. In the absence of infection, CF-TG cells constitutively exhibited an inflammatory signature, including genes that encode molecules such as IL-1alpha, IL-beta, IL-32, TNFSF14, LIF, CXCL1 and PLAU. In response to P. aeruginosa, genes associated with IFN-gamma response to infection (CXCL10, IL-24, IFNgammaR2) and other mediators of anti-infectious responses (CSF2, MMP1, MMP3, TLR2, S100 calcium-binding proteins A) were markedly up-regulated in wild-type TG cells. This microbicidal signature was silent in CF-TG cells. The deficiency of genes associated with IFN-gamma response was accompanied by the defective membrane expression of IFNgammaR2 and altered response of CF-TG cells to exogenous IFN-gamma. In addition, CF-TG cells were unable to secrete CXCL10, IL-24 and S100A8/S100A9 in response to P. aeruginosa. The differences between wild-type TG and CF-TG cells were due to the cftr mutation since gene expression was similar in wild-type TG cells and CF-TG cells transfected with a plasmid containing a functional cftr gene. Finally, we reported an altered sphingolipid metabolism in CF-TG cells, which may account for their inflammatory signature. This first comprehensive analysis of gene expression in TG cells proposes a protective role of wild-type TG against airborne pathogens and reveals an original program in which anti-infectious response was deficient in TG cells with a cftr mutation. This defective response may explain why host response does not contribute to protection against P. aeruginosa in CF.

The role of HCO(3)(-) transport in relation to fluid secretion by submucosal glands is being studied in sheep, pigs, cats and humans. Optical methods have been developed to measure secretion rates of mucus volume from single glands with sufficient temporal resolution to detect differences in minute-by-minute secretion rates among glands. The ionic composition and viscoelastic properties of the uncontaminated gland mucus are measured with a combination of ratiometric fluorescent indicators, ion-selective microelectrodes, FRAP, and a miniaturized, magnetic force viscometer. Sheep glands secreted basally at low rates, showed small, transient responses to alpha- and beta-adrenergic agonists, and large responses to a cholinergic agonist, carbachol. Peak rates and temporal patterns of responses to carbachol differed markedly among glands. To assess the contribution of HCO(3)(-) transport to gland secretion, we either inhibited Na(+)/K(+)/2Cl(-) cotransporter (NKCC) with bumetanide or replaced HCO(3)(-) with HEPES and gassed with O(2). Bumetanide caused a small, non-significant inhibition of basal secretion, but removal of HCO(3)(-)/CO(2) significantly reduced basal secretion almost by half. Both bumetanide and removal of HCO(3)(-)/CO(2) reduced carbachol-stimulated secretion significantly, with HCO(3)(-) removal having the larger effect: a reduction to 33% of control (P<0.01). The remaining secretory response to carbachol was nearly eliminated by bumetanide. Sheep mucus pH measured with ion selective electrodes was about 0.4 log more acidic than the bath. In humans, we observed the same pattern of responses to agonists and antagonists as in sheep, and observed a mucus pH of 7.0 using 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF). We hypothesize that HCO(3)(-) transport is important in the formation of mucus secretion, but that most HCO(3)(-) is scavenged before the final mucus appears at the duct opening. Cystic fibrosis transmembrane conductance regulator's (CFTR) best understood function is as an anion channel, but increasing attention has been given to its role in HCO(3)(-) transport. By analogy with organ-specific CFTR effects on Cl(-) transport, it seems likely that the relative importance of CFTR in HCO(3)(-) transport will also vary across organs. Because lung disease is by far the greatest cause of mortality among people with cystic fibrosis, it is important to determine how loss of CFTR function causes lung disease. We are testing the hypothesis that loss of CFTR alters serous cell secretion in the lungs, and the corollary that such loss contributes to cystic fibrosis (CF) lung disease. CFTR is highly expressed in serous cells of submucosal glands and the Calu-3 serous cell model secretes HCO(3)(-). Human gland serous cells grown in culture and tested for fluid secretion under open circuit conditions showed reduced fluid secretion to all mediators. However, submucosal glands are complex organs containing at least 4 distinct regions and at least that many cell types, making it difficult to predict the consequences on whole-organ function from experiments with individual cell types. Therefore, we have resurrected long-neglected methods for studying whole-gland function, and have attempted to improve them in a variety of ways. We are refining these methods and increasing our understanding of gland function by studying tracheal glands from sheep, pigs and cats. As human tissues become available, they are studied with the best methods presently available. The key questions now being asked are: Is mucus secretion from submucosal glands altered in cystic fibrosis? If so, how is it altered and how does it contribute to CF lung disease? Answering the last question will require an understanding of how glands interact with other regions of the lung. In the context of this meeting, we present preliminary data on the role of HCO(3)(-) in gland mucus secretion.

We sought an explanation for the accumulation, and apparent poor degradation by alveolar phagocytes, of alginate in cystic fibrosis lung. A crude intracellular lyase preparation extracted from Klebsiella pneumoniae was able to degrade seaweed alginic acid as well as forms purified from Pseudomonas aeruginosa bacteria from Cystic Fibrosis (CF) patient lungs. This was by a beta-eliminative mechanism, as detected by an increase in the 232nm absorbance and activity was enhanced by deacetylation of the Pseudomonas aeruginosa alginates. Conditioned media or cell lysates from unstimulated or triggered phagocytic cells (including resident mouse peritoneal macrophages and the human macrophage cell line U937) had no effect in the same system. Free radicals generated by chemical systems or by gamma irradiation of water degraded alginate. Depolymerisation by free radicals, as detected by viscosity determinations and polyacrylamide gel electrophoresis, generated a wide range of fragment sizes. In contrast, mouse peritoneal macrophages or human polymorphonuclear neutrophils stimulated to generate free radicals had no significant effect on alginate. Under the conditions of our experiments, phagocytic cells representative of the CF lung are not able to degrade Pseudomonas aeruginosa alginate. This may explain the gross accumulation of alginate in CF lung.

Several reports have indicated that fecal elastase-1 (EL-1) determination is a new, sensitive, and specific noninvasive pancreatic function test; however, very few patients with malabsorption due to small intestine diseases have been included in the previous studies. The aim of the study was to compare the diagnostic accuracy of fecal EL-1 and fecal chymotrypsin (FCT) in distinguishing between pancreatic maldigestion and intestinal malabsorption. Three groups of subjects were studied: group A included 49 patients with known cystic fibrosis (25 males, median age 5 years); group B included 43 subjects with various small intestine diseases (17 males, median age 6 years); and group C included 45 children without any history of gastrointestinal disease (22 males, median age 5 years). In all patients, stools were collected for 72 h on a standard diet and fecal EL-1, FCT, and steatocrit tests were performed. Both EL-1 and FCT were below normal limits in all CF patients with pancreatic maldigestion not treated with pancreatic enzyme (100% sensitivity for both assays); El-1, but not FCT, was also below normal in all the CF patients with pancreatic maldigestion treated with pancreatic extracts. Both EL-1 and FCT values in the CF group were significantly lower than in subjects with various small intestinal diseases and in children without any history of gastrointestinal disease (P < 0.0001). FCT, but not EL-1, values showed an inverse statistically significant correlation with steatocrit values in the whole CF group (P < 0.001); FCT was below normal in three of four CF patients with steatorrhea on pancreatic enzyme therapy. Both EL-1 and FCT had 100% specificity when calculated in children without any history of gastrointestinal disease; in contrast, specificity was 86% for EL-1 and 76% for FCT if we considered the control group with small intestinal diseases: low EL-1 was observed in two cases of intestinal giardiasis, two cases of short bowel syndrome, one case of celiac disease, and one case of intestinal pseudobstruction; FCT was abnormal in four cases of intestinal giardiasis, three cases of celiac disease, one case of short bowel syndrome, one case of Crohn's disease, and one case of intestinal pseudobstruction. Diagnostic accuracy was 92% for fecal EL-1 and 82% for FCT. Steatocrit values were over the normal limit in 11 patients with small intestine diseases; in 7/11 of these patients at least one of the pancreatic test results was below the normal limit. In conclusions, in patients with CF, fecal EL-1 determination is not more sensitive than FCT in identifying pancreatic maldigestion; however, fecal EL-1 assay is more specific than FCT determination in distinguishing pancreatic maldigestion from intestinal malabsorption.

The carotenoids are potent antioxidants with the ability to quench singlet oxygen and other toxic oxygen species. We studied 17 patients with cystic fibrosis (CF) and 10 normal children to assess plasma levels of four carotenoids, beta-carotene, alpha-carotene, lutein, and lycopene, by high-performance liquid chromatography. We found significantly lower plasma levels of specific carotenoids in children with CF than in normal control subjects. The standardization of carotenoid levels for total cholesterol did not significantly attenuate these differences. No differences in total carotene intake were apparent between the groups. Carotenoid levels did not correlate with fat absorption or measures of adiposity in children with CF. Additionally, levels of selected carotenoids correlated negatively with serum IgG levels, an indirect measure of inflammation. The differences in plasma carotenoid levels between children with CF and normal children may be due to rapid turnover of carotenoids, perhaps through quenching of toxic oxygen species in inflammatory states of CF. Studies assessing supplementation of these antioxidants should be considered.