OBJECTIVE

Wnt signaling pathway significantly participates in cardiac fibrosis and CFs activation. Therefore, we reviewed current evidence on the new perspectives and biological association between Wnt signaling pathway and cardiac fibrosis.

METHODS

A PubMed database search was performed for studies of Wnt signaling pathway in cardiac fibrosis and CFs activation.

RESULTS

Numerous studies have shown that the Wnt signaling pathway significantly participates in cardiac fibrosis pathogenesis. The aim of this review is to describe the present knowledge about the Wnt signaling pathway significantly participating in cardiac fibrosis and CFs activation, and look ahead on new perspectives of Wnt signaling pathway research. Moreover, we will discuss the different insights that interact with the Wnt signaling pathway-regulated cardiac fibrosis. The Wnt proteins are glycoproteins that bind to the Fz receptors on the cell surface, which lead to several important biological functions, such as cell differentiation and proliferation. There are several signals among the characterized pathways of cardiac fibrosis, including Wnt/β-catenin signaling. In this review, new insight into the Wnt signaling pathway in cardiac fibrosis pathogenesis is discussed, with special emphasis on Wnt/β-catenin.

CONCLUSIONS

It seems reasonable to suggest the potential targets of Wnt signaling pathway and it can be developed as a therapeutic target for cardiac fibrosis.

Antibacterials play a central role in the medical management of patients with cystic fibrosis (CF). Administration of adequate dosages of antibacterials results in pronounced beneficial effects on the morbidity and mortality of this patient group. The dosage of the antibacterial that is needed for optimal treatment depends on the individual patient's pharmacokinetics and the pharmacokinetic-pharmacodynamic effect on the micro-organism of relevance in the host. In general, the disposition of antibacterial drugs in patients with CF is not as 'atypical' as once thought. Recent research with adequately matched controls demonstrated that, for a few beta-lactam antibacterials only, a CF-specific increase of the total body clearance seems to exist and that the large volumes of distribution observed are the result of malnutrition and the relative lack of adipose tissue. Pharmacokinetic-pharmacodynamic relationships in patients with CF are less well studied. Apart from the pharmacokinetics, there is a need for optimisation of antibacterial therapy. For the aminoglycosides, pharmacokinetic optimisation based on measured serum drug concentrations is common practice. The Sawchuk-Zaske method based on peak and trough drug concentrations is widely used. A more sophisticated approach is the 'goal-oriented model-based Bayesian adaptive control' method, where integration of mathematically determined optimally (D-optimally) sampled serum drug concentrations and a population model results in the most likely set of individual pharmacokinetic parameter values suitable for further pharmacokinetic optimisation of the therapy. A future development is the integration of changing serum drug concentrations and killing rates of the target micro-organism to a pharmacokinetic-pharmacodynamic surrogate relationship to optimise drug therapy. The latter approach may be extremely useful in deciding on the frequency of aminoglycoside administration as well as the optimal use of the beta-lactam antibacterials and fluoroquinolones.

Forty patients with cystic fibrosis (CF), including 34 who died above age 10 years without having developed clinical diabetes mellitus and 6 who died with both cystic fibrosis and diabetes mellitus, were studied. The mean age of the female patients with CF but not diabetes was 15.8 +/- 5.6 years; of males without diabetes, 17.2 +/- 6.4 years; of female patients with CF and diabetes mellitus, 20.2 +/- 6.9 years; and of males with CF and diabetes, 21.3 +/- 6.6 years. The mean number of pancreatic islets in microscopic sections for patients with cystic fibrosis but not diabetes was 4.18 +/- 2.76/mm2, and the value for patients with both cystic fibrosis and diabetes mellitus was 2.61 +/- 2.07/mm2. The lowest density of pancreatic islets (1.69 +/- 0.48/mm2) for cystic fibrosis was found in patients with the latest-stage pathologic lesion. Nesidioblastosis (presence of ductuloinsular complexes) was identified in 14 of 38 cystic fibrosis patients, both with and without diabetes mellitus. The pancreatic islets of both diabetic and nondiabetic patients with CF showed hypertrophy; the mean volume of the three largest pancreatic islets for CF only was 0.0117 +/- 0.00657 mm3 and that for cystic fibrosis and diabetes was 0.00795 +/- 0.00599 mm3, both values being larger than normal. Ratios of the amounts of islet endocrine cells, A cells, B cells, and D cells, were determined by peroxidase--anti-peroxidase labeled antibody staining. The B cells composed 43.0% of endocrine cell mass in cystic fibrosis alone and 30.1% in cystic fibrosis with diabetes mellitus, which were lower than normal proportions. The D cell values, 11.9% in cystic fibrosis and 15.1% in cystic fibrosis with diabetes mellitus, on the other hand, were greater than normal ratios.

The pancreases of 23 patients (mean age 10.5 years, range 5-22) years dying of cystic fibrosis (CF) were evaluated at autopsy by routine histology and immunostaining for changes in their endocrine cell compartment. The severely altered pancreatic tissues showed end stage CF, with either a fibrotic pattern (CF-FIB, n = 14) or a lipoatrophic pattern (CF-LIP, n = 9) prevailing. In all specimens, irrespective of the dominating pattern, the islet system was affected by marked periinsular and intrainsular sclerosis. Quantitatively, the volume densities (relative tissue components) of the parenchymal, fibrotic, fatty and total endocrine compartments as well as the four islet cell types (B, A, D, PP) were determined by point counting. Compared with controls, the CF patients (including two patients with overt diabetes and glucose intolerance, respectively) had a significantly decreased insulin (B)-cell ratio (from 64.4 to 34%) with a concomitant rise in non-B-cells (A-cells: 23.2 to 35%; D-cells: 10.4 to 22%; PP-cells; 2 to 9%). Comparison of endocrine cell ratios in CF-FIB pancreases with CF-LIP pancreases revealed no significant differences. The reduction of approximately 50% of insulin cells in CF patients with advanced disease supports the concept that destruction of exocrine tissue with concomitant fibrous disorganization of islets gradually changes the proportional distribution of the endocrine cells in favor of the noninsulin cells. This slowly ongoing process probably provides the basis for islet dysfunction, i.e. diabetes, increasingly observed in final stage CF.

Cystic fibrosis (CF) is an autosomal recessive disorder of Cl(-) and Na(+) transport. The vast majority of CF patients have deleterious mutations in an epithelial Cl(-) channel called the CF transmembrane conductance regulator (CFTR). In contrast, defects in the epithelial Na(+) channel (SCNN1) have been associated with phenotypes dominated by renal disease (systemic pseudohypoaldosteronism type I and Liddle syndrome). We report two non-classic CF patients without CFTR mutations who have novel deleterious mutations in the beta-subunits of SCNN1 in the absence of overt renal disease.

Epigenetic changes through altered DNA methylation have been implicated in critical aspects of tumor progression, and have been extensively studied in a variety of cancer types. In contrast, our current knowledge of the aberrant genomic DNA methylation in tumor-associated fibroblasts (TAFs) or other stromal cells that act as critical coconspirators of tumor progression is very scarce. To address this gap of knowledge, we conducted genome-wide DNA methylation profiling on lung TAFs and paired control fibroblasts (<em>CFs</em>) from non-small cell lung cancer patients using the HumanMethylation450 microarray. We found widespread DNA hypomethylation concomitant with focal gain of DNA methylation in TAFs compared to <em>CFs</em>. The aberrant DNA methylation landscape of TAFs had a global impact on gene expression and a selective impact on the TGF-<em>β</em> pathway. The latter included promoter hypermethylation-associated SMAD3 silencing, which was associated with hyperresponsiveness to exogenous TGF-<em>β</em>1 in terms of contractility and extracellular matrix deposition. In turn, activation of <em>CFs</em> with exogenous TGF-<em>β</em>1 partially mimicked the epigenetic alterations observed in TAFs, suggesting that TGF-<em>β</em>1 may be necessary but not sufficient to elicit such alterations. Moreover, integrated pathway-enrichment analyses of the DNA methylation alterations revealed that a fraction of TAFs may be bone marrow-derived fibrocytes. Finally, survival analyses using DNA methylation and gene expression datasets identified aberrant DNA methylation on the EDARADD promoter sequence as a prognostic factor in non-small cell lung cancer patients. Our findings shed light on the unique origin and molecular alterations underlying the aberrant phenotype of lung TAFs, and identify a stromal biomarker with potential clinical relevance.

BACKGROUND

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is accompanied by a) systemic IgA/IgM responses against the lipopolysaccharides (LPS) of commensal bacteria; b) inflammation, e.g. increased plasma interleukin-(IL)1 and tumor necrosis factor (TNF)α; and c) activation of cell-mediated immunity (CMI), as demonstrated by increased neopterin.

METHODS

To study the relationships between the IgA/IgM responses to the LPS of microbiota, inflammation, CMI and the symptoms of ME/CFS we measured the IgA/IgM responses to the LPS of 6 different enterobacteria, serum IL-1, TNFα, neopterin, and elastase in 128 patients with ME/CFS and chronic fatigue (CF). Severity of symptoms was assessed by the Fibromyalgia and Chronic Fatigue Syndrome (FF) Rating Scale.

RESULTS

Serum IL-1, TNFα, neopterin and elastase are significantly higher in patients with ME/CFS than in CF patients. There are significant and positive associations between the IgA responses to LPS and serum IL-1, TNFα, neopterin and elastase. Patients with an abnormally high IgA response show increased serum IL-1, TNFα and neopterin levels, and higher ratings on irritable bowel syndrome (IBS) than subjects with a normal IgA response. Serum IL-1, TNFα and neopterin are significantly related to fatigue, a flu-like malaise, autonomic symptoms, neurocognitive disorders, sadness and irritability.

CONCLUSIONS

The findings show that increased IgA responses to commensal bacteria in ME/CFS are associated with inflammation and CMI activation, which are associated with symptom severity. It is concluded that increased translocation of commensal bacteria may be responsible for the disease activity in some ME/CFS patients.

To assess the type, severity, and regional lung distribution of cystic fibrosis (CF) lesions as shown using high-resolution CT (HRCT), comparing these findings with chest radiographs and pulmonary function tests (PFTs), we obtained HRCTs in 36 patients with CF (mean age 13), who were clinically stable. We assessed four lung regions (upper and lower, right and left) and assigned each a semiquantitative score for (a) bronchial abnormalities, (b) parenchymal abnormalities, and (c) overinflation, based on the severity and profusion of the corresponding lesions. A similar regional assessment of chest radiographs was also done using the Chrispin-Norman method. PFT results were correlated with the radiological data. On HRCT, bronchial lesions were present in 89% of the patients and in 78% of the regions; bronchiectasis was the predominant abnormality in our population, visible in 100% of the abnormal regions. Less frequent were bronchial wall thickening (48%) and mucous plugs (29%). Parenchymal abnormalities were recognizable in 58% of the patients and 31% of the regions; alveolar consolidation was more frequent (80%) than were destructive changes (36%). Overinflation was found in 81% of the patients and 85% of the regions. We found the severity and profusion of bronchial lesions and parenchymal destructive changes to be unevenly distributed among the different regions, the upper lungs being more heavily involved than the lower, particularly on the right. Alveolar consolidation and overinflation were more uniform in distribution. HRCT patient scores correlated significantly with radiographic scores (r = 0.861) and with PFTs, especially with forced expiratory volume for 1 s (FEV1; r = 0.658). HRCT can be useful in the clinical management of patients with CF, depicting the type and distribution of bronchial and parenchymal lesions, particularly when chest radiographic results are unclear. In the planning and postural drainage, special attention should be given to the apical and posterior parts of the lungs, especially on the right; these are the areas most frequently and most severely involved by the disease.

Studies of the prevalence of Burkholderia cepacia complex species amongst cystic fibrosis (CF) patients in different geographical regions, and the association between cross-infection and putative transmissibility markers, will further our understanding of these organisms and help to address infection-control issues. In this study, B. cepacia complex isolates from CF patients in different regions of Europe were analysed. Isolates were examined for B. cepacia complex species and putative transmissibility markers [cable pilin subunit gene (cblA) and the B. cepacia epidemic strain marker (BCESM)]. Sporadic and cross-infective strains were identified by random amplification of polymorphic DNA (RAPD). In total, 79% of patients were infected with Burkholderia cenocepacia (genomovar III), 18% with Burkholderia multivorans (genomovar II) and less than 5% of patients with B. cepacia (genomovar I), Burkholderia stabilis (genomovar IV) or Burkholderia vietnamiensis (genomovar V). The cblA and BCESM transmissibility markers were only detected in strains of B. cenocepacia. The BCESM was a more sensitive marker for transmissible B. cenocepacia strains than cblA, although sporadic B. cenocepacia strains containing the BCESM, but lacking cblA, were also observed. Furthermore, clusters of cross-infection with transmissibility marker-negative strains of B. multivorans were identified. In conclusion, B. cenocepacia was the greatest cause of cross-infection, and the most widely distributed B. cepacia complex species, within these CF populations. However, cross-infection was not exclusive to B. cenocepacia and cblA and the BCESM were not absolute markers for transmissible B. cenocepacia, or other B. cepacia complex strains. It is therefore suggested that CF centres cohort patients based on the presence or absence of B. cepacia complex infection and not on the basis of transmissibility marker-positive B. cenocepacia as previously suggested.

One or more of five morphologically distinct classes of appendage pili were determined to be peritrichously expressed by Burkholderia (formerly Pseudomonas) cepacia isolated from disparate sources. B. cepacia-encoded cblA pilin gene hybridization-based analysis revealed that one associated class, cable (Cbl) adhesin type IIB. cepacia pili, correlates with epidemically transmitted strains from a single cystic fibrosis (CF) center. When only phenotypic assays were available, correlations between the source and the pilus type were nonetheless observed: filamentous (Fil) type IIIB. cepacia pili correlated with CF-associated nonepidemic isolates, spine (Spn) type IVB. cepacia pili correlated with clinical (non-CF) isolates, and spike (Spk) type VB. cepacia pili correlated with environmental isolates. Further, Cbl, Fil, or Spk pili typically appear as an internal framework for constitutively coexpressed, peritrichously arranged dense mats of fine, curly mesh (Msh) type IB. cepacia pili. Constitutive coexpression of dense mats of Msh type IB. cepacia pili in association with a labyrinth of either Cbl, Fil, or Spk pili suggests possible cooperative pilus interactions mediating adhesion-based colonization in the differing environments from which the strains were isolated. Despite such correlations, phylogenetic analyses indicate that with the exception of the epidemically transmitted clusters of isolates, the remaining B. cepacia strains from the other three sources exhibited an equal degree of genetic relatedness independent of origin. As previously found for Escherichia coli, this discrepancy could be accounted for by selection-driven, in vivo horizontal transfer events between distantly related members of the species B. cepacia, leading to the genetic acquisition of environmentally appropriate adhesion-based colonization pilus operons.

It was shown earlier that immune responses against cholera toxin (CT) as well as Vibrio cholerae lipopolysaccharide (LPS) or whole bacterial cells (WC) were protective and that these different antibody specificities co-operated synergistically for protection against experimental cholera. Similarly, antibodies against the heat-labile toxin (LT) and major colonization factors (CFs) of enterotoxingenic Escherichia coli (ETEC) co-operated synergistically for protection against LT-producing ETEC expressing homologous CFs. Studies in humans revealed that repeated oral antigen administration was optimal in inducing intestinal immune responses. Based on these findings oral inactivated vaccines consisting of toxin antigen and whole cells, i.e. the licensed recombinant cholera B subunit (rCTB)-WC cholera vaccine Dukoral®, and candidate ETEC vaccines have been developed. In different trials the rCTB-WC cholera vaccine has provided very high (85-100%) short term protection, which was significantly higher than that induced by the WC component alone, whereas rCTB-WC and WC alone provided comparable (50-60%), long term protection. An oral ETEC vaccine consisting of rCTB and formalin-inactivated E. coli bacteria expressing major CFs was shown to be safe and immunogenic in adults and children in different countries. The vaccine also induced significant protection against non-mild ETEC diarrhoea, i.e. diarrhoea interfering with daily activity in American travellers but not against ETEC diarrhoea in young children in Egypt. Against this background, a modified ETEC vaccine consisting of recombinant E. coli strains overexpressing the major CFs and a more LT like hybrid toxoid (LCTBA) has been developed. This vaccine will be tested soon alone and together with a mucosal adjuvant, i.e. dmLT, in clinical trials.

Hypermutable bacterial pathogens exist at surprisingly high prevalence and benefit bacterial populations by promoting adaptation to selective environments, including resistance to antibiotics. Five hundred Haemophilus influenzae isolates were screened for an increased frequency of mutation to resistance to rifampicin, nalidixic acid and spectinomycin: of the 14 hypermutable isolates identified, 12 were isolated from cystic fibrosis (CF) sputum. Analysis by enterobacterial repetitive intergenic consensus (ERIC)-PCR and ribotyping identified eight distinct genetic fingerprints. The hypermutable phenotype of seven of the eight unique isolates was associated with polymorphisms in conserved sites of mutS. Four of the mutant mutS alleles were cloned and failed to complement the mutator phenotype of a mutS : : TSTE mutant of H. influenzae strain Rd KW20. Antibiotic susceptibility testing of the hypermutators identified one beta-lactamase-negative ampicillin-resistant (BLNAR) isolate with two isolates producing beta-lactamase. Six isolates from the same patient with CF, with the same genetic fingerprint, were clonal by multilocus sequence typing (MLST). In this clone, there was an evolution to higher MIC values for the antibiotics administered to the patient during the period in which the strains were isolated. Hypermutable H. influenzae with mutations in mutS are prevalent, particularly in the CF lung environment, and may be selected for and maintained by antibiotic pressure.

Between the 3rd and 6th day post coitum the dry substance of uterine fluid contains nearly 60% peptide chains, on the 6th day 19 h p.c. only 35%. The remaining 40 and 65% resp. consist mainly of carbohydrates (cf. III.1). By means of disc electrophoresis, thin layer chromatography following hydrolysis and molecular sieve chromatography, we were able to characterize 4 uterine specific proteins, 2 of which are carbohydrate rich glycoproteins. Their relative concentrations were determined between 0 and 8 day p.c. (of. III.2): Glycoprotein I can already be found during oestrus. Its concentration increases slightly from the 3rd to the 5th day p.c., more rapidly up to the 6th day 6 h p.c., and decreases later (of.III.2b). The molecular weight is estimated to be around 50,000 (cf.III.4). Postalbumin as well as Uteroglobin can be found shortly after ovulation. Its concentration stays nearly constant between 3rd and 6th day p.c., and decreases during the 6th day p.c. (of.III.2b). Its molecular weight is in the same range as that of Uteroglobin, nearly 30,000 (of.III.4. Uteroglobin is most probably free of carbohydrates. Its concentration increases rapidly between 16 and 40 h p.c., slightly until the 5th day p.c., and decreases later (cf.III.2b). At 6th day p.c. about 50% of the peptide chains of uterine fluid are Uteroglobin (cf.III.2.g). Glycoprotein II is not secreted before the 6th day p.c. It has a comparativly high content of galactose (cf.III.3.d). Its concentration increases rapidly during the 6th day p.c. (cf.III.2.b). The moleculare weight is higher than 70,000 (cf.III. 4).These proteins and glycoproteins have also been found inblastocoelic fluid after the 5th day p.c. (cf.III.2.c).Aftersuperovulation induced by hormones, and natural mating, the pregnancy specific protein pattern is not fully developed (cf.III.2.d). Inpseudopregnancy, triggered by mating with vasectomized males, the same proteins as in normal pregnancy were found from the 6th day 6 h to the 6th day 19 h p.c. If no blastocysts are implanted, the protein pattern of the 6th day 19 h p.c. persists nearly unchanged until the 9th day p.c. (cf.III.2.e).Duringimplantation uterus and blastocyst lumens are filled with serum proteins, which tend to cover up the secretion proteins (cf.III.2.f).9 constituents of carbohydrates could be identified in uterine fluid. There was no evidence for freemonohexoses, particularly free glucose. Theoligomere carbohydrates were free or bound to proteins (cf.III.3.). We were able to separate Glycoprotein I, Glycoprotein II, Albumin + Uteroglobin with Sephadex G 150 and G 75 SF (cf.III.4).21 freeamino acids were found in uterine and blastocoelic fluid by means of thin layer chromatography. The concentrations of amino acids in uterine fluid were comparable to those of serum. The dry substance of uterine fluid contains more glycine, alanine, glutamic acid and aspartic acid than that of serum (cf.III.5).The possible role of amino acids in thenutrition of the blastocyst and the possible influence of uterine specific proteins and glycoproteins onnutrition and morphogenesis of the rabbit embryo were discussed.

OBJECTIVE

With their potent activity against Gram-negative bacteria, the polymyxins are important alternative antibiotics for cystic fibrosis (CF) patients. A retrospective evaluation of polymyxin activity against 6001 Pseudomonas aeruginosa, 150 Achromobacter xylosoxidans and 506 Stenotrophomonas maltophilia CF isolates was initiated. In addition, we looked at how polymyxin susceptibility testing was affected by the testing method (agar dilution versus microdilution), the agent (polymyxin E versus polymyxin B), incubation time (24 h versus 48 h) and by different interpretative criteria (German DIN, French FSM, British BSAC).

RESULTS

Polymyxin B exhibited reasonable activity against P. aeruginosa (MIC(90)< or =2 mg/L), whereas it was less active against A. xylosoxidans (MIC(90)< or =16 mg/L) and S. maltophilia (MIC(90)< or =16 mg/L). During 2000-2002, polymyxin B resistance in P. aeruginosa, S. maltophilia and A. xylosoxidans was found to be 6.7%, 17.0% and 29.9% (corresponding to 12.4%, 20.7% and 35.4% of infected patients), respectively. When the agar dilution method was used, polymyxin E exhibited higher MICs than polymyxin B. The microdilution method produced lower polymyxin MICs than the agar dilution method. Therefore, the microdilution MICs after prolonged incubation (48 h) and the agar dilution MICs of polymyxin B correlated best (AUC of 0.93, r(2) of 0.44 and s of 0.83).

CONCLUSIONS

Polymyxin resistance among common CF pathogens is not rare, thus underlining the necessity of accurate susceptibility testing. When compared with the agar dilution method, it was found that the microdilution method is a valid, rapid and cost effective alternative for the determination of polymyxin activity. The performance of the microdilution method was most reliable after prolonged incubation (48 h) at a susceptibility breakpoint of < or =4 mg/L according to the BSAC guidelines (specificity 91%, sensitivity 89%, 1.5% very major errors).

Immune responses against colonization factors (CFs) and the nontoxic B component of the enterotoxigenic Escherichia coli (ETEC) heat-labile toxin (LTB) are considered to be important for immunity against diarrhea caused by ETEC. Individual live attenuated ETEC derivatives that have had their toxin genes removed and whose aroC, ompC, and ompF genes are deleted have shown promise as vaccines against ETEC. The development of such strains has culminated in the testing of a three-strain-combination live attenuated vaccine known as ACE527, comprised of strains ACAM2025 expressing colonization factor antigen I (CFA/I) and LTB; ACAM2022, expressing CS5, CS6, and LTB; and ACAM2027, expressing CS1, CS2, CS3, and LTB. The recombinant CF and LTB genes expressed in the three strains were inserted into the bacterial chromosome to ensure their stable inheritance and expression without the requirement for any selection. ACE527 has been tested in a randomized placebo-controlled, double-blind, phase I safety and immunogenicity study in healthy adult volunteers and proved to be well tolerated and immunogenic at dose levels of 10(10) and 10(11) total CFU. There was no indication of strain interference on the basis of fecal shedding patterns, with all three being detected in the feces of 50% and 83% of low- and high-dose vaccine recipients, respectively. Similarly, strong immune responses to LTB and to CFs expressed on all three constituent strains were induced, with at least 50% of subjects in the high-dose group responding to LTB, CFA/I, CS3, and CS6.

Deletion of F508 in the first nucleotide binding domain (NBD1) of cystic fibrosis transmembrane conductance regulator protein (CFTR) is the commonest cause of cystic fibrosis (CF). Functional interactions between CFTR and CK2, a highly pleiotropic protein kinase, have been recently described which are perturbed by the F508 deletion. Here we show that both NBD1 wild type and NBD1 DeltaF508 are phosphorylated in vitro by CK2 catalytic alpha-subunit but not by CK2 holoenzyme unless polylysine is added. MS analysis reveals that, in both NBD1 wild type and DeltaF508, the phosphorylated residues are S422 and S670, while phosphorylation of S511 could not be detected. Accordingly, peptides encompassing the 500-518 sequence of CFTR are not phosphorylated by CK2; rather they inhibit CK2alpha catalytic activity in a manner which is not competitive with respect to the specific CK2 peptide substrate. In contrast, 500-518 peptides promote the phosphorylation of NBD1 by CK2 holoenzyme overcoming inhibition by the beta-subunit. Such a stimulatory efficacy of the CFTR 500-518 peptide is dramatically enhanced by deletion of F508 and is abolished by deletion of the II507 doublet. Kinetics of NBD1 phosphorylation by CK2 holoenzyme, but not by CK2alpha, display a sigmoid shape denoting a positive cooperativity which is dramatically enhanced by the addition of the DeltaF508 CFTR peptide. SPR analysis shows that NBD1 DeltaF508 interacts more tightly than NBD1 wt with the alpha-subunit of CK2 and that CFTR peptides which are able to trigger NBD1 phosphorylation by CK2 holoenzyme also perturb the interaction between the alpha- and the beta-subunits of CK2.

OBJECTIVE

Chronic fatigue syndrome (CFS) and multiple sclerosis (MS) are characterized by debilitating fatigue, yet evaluation of this symptom is subjective. We examined metabolite-detecting, adrenergic, and immune gene expression (messenger ribonucleic acid [mRNA]) in patients with CFS (n = 22) versus patients with MS (n = 20) versus healthy controls (n = 23) and determined their relationship to fatigue and pain before and after exercise.

METHODS

Blood samples and fatigue and pain ratings were obtained at baseline and 0.5, 8, 24, and 48 hours after sustained moderate exercise. Leukocyte mRNA of four metabolite-detecting receptors (acid-sensing ion channel 3, purinergic type 2X4 and 2X5 receptors, and transient receptor potential vanilloid type 1) and four adrenergic (α-2a, β-1, and β-2 receptors and catechol-O-methyltransferase) and five immune markers (CD14, toll-like receptor 4 [TLR4], interleukin [IL] 6, IL-10, and lymphotoxin α) was examined using quantitative polymerase chain reaction.

RESULTS

Patients with CFS had greater postexercise increases in fatigue and pain (10-29 points above baseline, p < .001) and greater mRNA increases in purinergic type 2X4 receptor, transient receptor potential vanilloid type 1, CD14, and all adrenergic receptors than controls (mean ± standard error = 1.3 ± 0.14- to 3.4 ± 0.90-fold increase above baseline, p = .04-.005). Patients with CFS with comorbid fibromyalgia (n = 18) also showed greater increases in acid-sensing ion channel 3 and purinergic type 2X5 receptors (p < .05). Patients with MS had greater postexercise increases than controls in β-1 and β-2 adrenergic receptor expressions (1.4 ± 0.27- and 1.3 ± 0.06-fold increases, respectively, p = .02 and p < .001) and greater decreases in TLR4 (p = .02). In MS, IL-10 and TLR4 decreases correlated with higher fatigue scores.

CONCLUSIONS

Postexercise mRNA increases in metabolite-detecting receptors were unique to patients with CFS, whereas both patients with MS and patients with CFS showed abnormal increases in adrenergic receptors. Among patients with MS, greater fatigue was correlated with blunted immune marker expression.

Intracellular ion homeostasis in liver cell is accomplished through the concerted action of ion pumps, carriers and channels. Description of these elements includes the Na+/K+-ATPase, Na+/H+- and Cl-/ HCO3(-)-exchange and Na+-HCO-3(-)- and Na(+)-K(+)-2 Cl(-)-cotransport as well as K+-, Cl(-)-, Na+ and nonselective cation channels. A short section is devoted to regulation of intracellular Ca2+ concentration. Ion flux through these elements is regulated by hormones and during experimental alterations of cell volume. We describe the effects of hormones and agonists on ion transport (e.g. purinergic agonists, alpha- and beta-adrenergic agonists, vasopressin, glucagon and insulin) and we emphasize the fact that altered ion transport rates may lead to cell swelling or cell shrinkage. Osmotically induced cell swelling or cell shrinkage triggers volume regulatory responses: recovery from cell swelling is accomplished by release of K+ and Cl- through ion channels whereas volume gain following osmotic shrinkage involves uptake of Na+, K+ and Cl- by channels, carriers and the Na+/K+-pump. Osmotic cell volume changes are associated with a broad spectrum of altered cell functions that includes changes of bile flow and bile acid transport, modulation of cytoplasmic and endosomal pH, metabolism of carbohydrate, protein and lipids, transcription and translation and alteration of cytoskeleton components (cf. Häussinger & Schliess; J. Hepatology 1995; 22: 94-100). In conclusion, we stress the concept that transduction of a hormonal signal primarily involves alteration of membrane ion transport followed by a change in cell volume. This change in cell volume appears to assist in executing a hormonal stimulus on cell function.

At the Danish CF Center patients with chronic Pseudomonas aeruginosa lung infection were treated 3-4 times a year (from 1976) with a 2-week intravenous antipseudomonal course which included preferentially an aminoglycoside and a beta-lactam antibiotic. We investigated the development of antibiotic resistance in P. aeruginosa strains isolated from Danish CF patients over a period of 18 years by testing the in vitro efficacy of carbenicillin, piperacillin, ceftazidime, tobramycin and ciprofloxacin against P. aeruginosa strains collected in 1973 (51 strains), 1980 (80 strains), 1985 (58 strains), and 1991 (100 strains). All the strains were screened and assayed semiquantitatively for beta-lactamase activity by use of nitrocefin. We found a significant (p < 0.005) increase in the MIC values of the P. aeruginosa strains against piperacillin and ceftazidime. However, no significant correlation was found between the MIC and the number of antipseudomonal courses of antibiotics. The proportion of resistant in vivo selected P. aeruginosa strains, presumed to be stably derepressed producers of chromosomal beta-lactamase, also increased significantly during the period studied. Our results confirm that the beta-lactamase production is an important mechanism of antibiotic resistance in P. aeruginosa.

Boswellin (BE), a methanol extract of the gum resin exudate of Boswellia serrata, contains naturally occurring triterpenoids, beta-boswellic acid and its structural related derivatives, has been used as a traditional medicine for the treatment of inflammatory and arthritic diseases. Topical application of BE to the backs of mice markedly inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced increases in skin inflammation, epidermal proliferation, the number of epidermal cell layers, and tumor promotion in 7,12-dimethylbenz[a]anthracene (DMBA)-initiated mice. Feeding 0.2% of BE in the diet to CF-1 mice for 10-24 weeks reduced the accumulation of parametrial fat pad weight under the abdomen, and inhibited azoxymethane (AOM)-induced formation of aberrant crypt foci (ACF) by 46%. Addition of pure beta-boswellic acid, 3-O-acetyl-beta-boswellic acid, 11-keto-beta-boswellic acid or 3-O-acetyl-11-keto-beta-boswellic acid to human leukemia HL-60 cell culture inhibited DNA synthesis in HL-60 cells in a dose-dependent manner with IC50 values ranging from 0.6 to 7.1 microM. These results indicate that beta-boswellic acid and its derivatives (the major constituents of Boswellin) have anti-carcinogenic, anti-tumor, and anti-hyperlipidemic activities.

Despite being implicated as important intermediates, iron(V) compounds have proven very challenging to isolate and characterize. Here, we report the preparation of the iron(V) nitrido complex, [PhB((t)BuIm)(3)Fe(V)≡N]BAr(F24) (PhB((t)BuIm)(3)(-) = phenyltris(3-tert-butylimidazol-2-ylidene)borato, BAr(F24) = B(3,5-(CF(3))(2)C(6)H(3))(4)(-)), by one electron oxidation of the iron(IV) nitrido precursor. Single-crystal x-ray diffraction of the iron(V) complex reveals a four-coordinate metal ion with a terminal nitrido ligand. Mößbauer and electron paramagnetic resonance spectroscopic characterization, supported by electronic structure calculations, provide evidence for a d(3) iron(V) metal center in a low spin (S = 1/2) electron configuration. Low-temperature reaction of the iron(V) nitrido complex with water under reducing conditions leads to high yields of ammonia with concomitant formation of an iron(II) species.

BACKGROUND

The relationship between lower airway markers of inflammation and infection with physiologic findings is poorly understood in young children with cystic fibrosis (CF). The goal of this study was to evaluate the association of bronchoalveolar lavage fluid (BALF) markers of infection and inflammation, including mediators linked to airway remodeling, to infant lung function values in young children with CF undergoing clinically indicated bronchoscopy.

METHODS

Plethysmography and the raised volume rapid thoracoabdominal compression (RVRTC) technique were performed in 16 sedated infants and young children with CF prior to bronchoscopy. BALF was collected and analyzed for pathogen density, cell count, % neutrophils, transforming growth factor beta 1 (TGF-beta(1)), matrix metalloproteinases (MMP), and interleukin-8 (IL-8).

RESULTS

There was a significant direct correlation between functional residual capacity (FRC), the ratio of residual volume to total lung capacity (RV/TLC) and FRC/TLC with % neutrophils (P < 0.05). Forced expiratory flows were inversely correlated to % neutrophils (P < 0.01). Lung function parameters did not differentiate those with and without lower airway infection; however, pathogen density directly correlated with FRC and inversely correlated with flows (P < 0.05). In a subset of the population, MMP-2 directly correlated with RV/TLC and inversely correlated with flows (P < 0.05) and TGF-beta(1) directly correlated with FRC (P < 0.05).

CONCLUSIONS

Results from this study suggest that lower airway inflammation as well as mediators linked to airway remodeling play an active role in pulmonary deterioration in CF infants and young children undergoing clinically indicated bronchoscopy.

BACKGROUND

As Chronic Fatigue Syndrome (CFS) has been known to follow Epstein-Bar virus (EBV) and other systemic infections; our objective was to describe differences in immune activation in post-infective CFS (PI-CFS) patients and recovered controls. We studied 301 adolescents prospectively over 24 months following the diagnosis of monospot-positive infectious mononucleosis (IM). We found an incidence of CFS at 6, 12 and 24 months of 13%, 7% and 4% respectively.

METHODS

Using chemiluminescent imaging we measured the concentrations of IL-1a, 1b, 2, 4, 5, 6, 8, 10, 12 (p70), 13, 15, 17 and 23, IFN-γ, TNF-α and TNF-β in duplicate plasma samples available in bio-bank from 9 PI-CFS subjects and 12 recovered controls at 24 months post-infection.

RESULTS

Standard comparative analysis indicated significant differences in IL-8 and 23 across subject groups. In constructing a linear classification model IL-6, 8 and 23 were selected by two different statistical approaches as discriminating features, with IL-1a, IL-2 and IFN-γ also selected in one model or the other. This supported an assignment accuracy of better than 80% at a confidence level of 0.95 into PI-CFS versus recovered controls.

CONCLUSIONS

These results suggest that co-expression patterns in as few as 5 cytokines associated with Th17 function may hold promise as a tool for the diagnosis of post-infectious CFS.

The interior of chlorosomes of green bacteria forms an unusual antenna system organized without proteins. The steady-spectra (absorption, circular dichroism, and linear dichroism) have been modeled using the Frenkel Hamiltonian for the large tubular aggregates of bacteriochlorophylls with geometries corresponding to those proposed for Chloroflexus aurantiacus and Chlorobium tepidum chlorosomes. For the Cf. aurantiacus aggregates we apply a structure used previously (V. I. Prokhorenko., D. B. Steensgaard, and A. R. Holzwarth, Biophys: J. 2000, 79:2105-2120), whereas for the Cb. tepidum aggregates a new extended model of double-tube aggregates, based on recently published solid-state nuclear magnetic resonance studies (B.-J. van Rossum, B. Y. van Duhl, D. B. Steensgaard, T. S. Balaban, A. R. Holzwarth, K. Schaffner, and H. J. M. de Groot, Biochemistry 2001, 40:1587-1595), is developed. We find that the circular dichroism spectra depend strongly on the aggregate length for both types of chlorosomes. Their shape changes from "type-II" (negative at short wavelengths to positive at long wavelengths) to the "mixed-type" (negative-positive-negative) in the nomenclature proposed in K. Griebenow, A. R. Holzwarth, F. van Mourik, and R. van Grondelle, Biochim: Biophys. Acta 1991, 1058:194-202, for an aggregate length of 30-40 bacteriochlorophyll molecules per stack. This "size effect" on the circular dichroism spectra is caused by appearance of macroscopic chirality due to circular distribution of the transition dipole moment of the monomers. We visualize these distributions, and also the corresponding Frenkel excitons, using a novel presentation technique. The observed size effects provide a key to explain many previously puzzling and seemingly contradictory experimental data in the literature on the circular and linear dichroism spectra of seemingly identical types of chlorosomes.