Nearly every form of the heart disease is associated with myocardial fibrosis, which is characterized by the accumulation of activated cardiac fibroblasts (CFs) and excess deposition of extracellular matrix (ECM). Although, CFs are the primary mediators of myocardial fibrosis in a diseased heart, in the traditional view, activated CFs (myofibroblasts) and resulting fibrosis were simply considered the secondary consequence of the disease, not the cause. Recent studies from our lab and others have challenged this concept by demonstrating that fibroblast activation and fibrosis are not simply the secondary consequence of a diseased heart, but are crucial for mediating various myocardial disease processes. In regards to the mechanism, the vast majority of literature is focused on the direct role of canonical SMAD-2/3-mediated TGF-β signaling to govern the fibrogenic process. Herein, we will discuss the emerging role of the GSK-3β, β-catenin and TGF-βCF-specific genetic manipulation can lead to aberrant myocardial fibrosis and sturdy cardiac phenotype. This will allow for a better understanding of the driving role of CFs in the myocardial disease process. We will also review the specificity and limitations of the currently available genetic tools used to study myocardial fibrosis and its associated mechanisms. A better understanding of the GSK-3β, β-catenin and SMAD-3 signaling network may provide a novel therapeutic target for the management of myocardial fibrosis in the diseased heart.

Innate lymphoid cells (ILCs) are crucial for the immune surveillance at mucosal sites. ILCs coordinate early eradication of pathogens and contribute to tissue healing and remodeling, features that are dysfunctional in patients with cystic fibrosis (CF). The mechanisms by which ILCs contribute to CF-immunopathology are ill-defined. Here, we show that group 2 ILCs (ILC2s) transdifferentiated into IL-17-secreting cells in the presence of the epithelial-derived cytokines IL-1β, IL-23 and TGF-β. This conversion is abrogated by IL-4 or vitamin D3. IL-17 producing ILC2s induce IL-8 secretion by epithelial cells and their presence in nasal polyps of CF patients is associated with neutrophilia. Our data suggest that ILC2s undergo transdifferentiation in CF nasal polyps in response to local cytokines, which are induced by infectious agents.

OBJECTIVE

Recent literature on Aspergillus fumigatus infection and allergy in cystic fibrosis have expanded our understanding of many aspects of allergic bronchopulmonary aspergillosis, and bring new attention to A. fumigatus airways infection and A. fumigatus allergy without allergic bronchopulmonary aspergillosis (ABPA).

RESULTS

ABPA, A. fumigatus infection and A. fumigatus allergy without ABPA all likely worsen cystic fibrosis (CF) lung disease. Studies examining utility of new serologic assays for diagnosing ABPA include evaluations of standardized measurement of A. fumigatus-specific IgG, serum chemokine TARC levels, and recombinant A. fumigatus allergens; as yet, none appear ideal. Although oral glucocorticoids remain primary therapy, toxicity and incomplete control have led to an ongoing search for further safe and effective agents including itraconazole and voriconazole, intravenous pulse methylprednisolone, nebulized amphotericin B and omalizumab. Little controlled treatment data is available.

CONCLUSIONS

Diagnosis of CF-ABPA remains difficult, but improvements in serologic assays are occurring. Treatment remains in many cases unsatisfactory, and new agents offer promise but await proper controlled trials of safety and efficacy. A. fumigatus airway infection and A. fumigatus allergy without ABPA are emerging as further complications of A. fumigatus respiratory colonization in patients with CF, but prospective studies are needed to corroborate largely retrospective findings.

BACKGROUND

It is unclear which factors explain the high co-morbidity between functional dyspepsia (FD) and other functional somatic syndromes. The aim of this study is to investigate the association between gastric sensorimotor function, psychosocial factors and 'somatization' on the one hand, and co-morbid irritable bowel syndrome (IBS) and chronic fatigue (CF)-like symptoms on the other, in FD.

METHODS

In 259 tertiary care FD patients, we studied gastric sensorimotor function with barostat (sensitivity, accommodation). We measured psychosocial factors (abuse history, alexithymia, trait anxiety, depression, panic disorder) and 'somatization' using self-report questionnaires, and presence of IBS and CF-like symptoms. Hierarchical multiple logistic regression was used to determine which of these factors were independently associated with co-morbid IBS and CF-like symptoms, including testing of potential mediator effects.

RESULTS

Co-morbid IBS or CF-like symptoms respectively were found in 142 (56.8%) and 102 (39.4%) patients; both co-morbidities were not significantly associated (P=0.27). Gastric accommodation (β=0.003, P=0.04) and 'somatization' (β=0.17, P= 0.0003) were independent risk factors for IBS (c=0.74, P<0.0001); the effect of adult abuse (β=0.72, P=0.20) was mediated by 'somatization'. Depression (β=0.16, P=0.008) and 'somatization' (β=0.18, P=0.004) were overlapping risk factors for CF-like symptoms (c=0.83, P<0.0001); the effects of alexithymia and lifetime abuse were mediated by depression and 'somatization', respectively.

CONCLUSIONS

'Somatization' is a common risk factor for co-morbid IBS and CF-like symptoms in FD and mediates the effect of abuse. Gastric sensorimotor function and depression are specific risk factors for co-morbid IBS and CF-like symptoms, respectively.

BACKGROUND

Inflammation in the lung is the body's natural response to injury. It acts to remove harmful stimuli such as pathogens, irritants, and damaged cells and initiate the healing process. Acute and chronic pulmonary inflammation are seen in different respiratory diseases such as; acute respiratory distress syndrome, chronic obstructive pulmonary disease (COPD), asthma, and cystic fibrosis (CF).

RESULTS

In this review, we found that inflammatory response in COPD is determined by the activation of epithelial cells and macrophages in the respiratory tract. Epithelial cells and macrophages discharge transforming growth factor-β (TGF-β), which trigger fibroblast proliferation and tissue remodeling. Asthma leads to airway hyper-responsiveness, obstruction, mucus hyper-production, and airway-wall remodeling. Cytokines, allergens, chemokines, and infectious agents are the main stimuli that activate signaling pathways in epithelial cells in asthma. Mutation of the CF transmembrane conductance regulator (CFTR) gene results in CF. Mutations in CFTR influence the lung epithelial innate immune function that leads to exaggerated and ineffective airway inflammation that fails to abolish pulmonary pathogens. We present mechanistic computational models (based on ordinary differential equations, partial differential equations and agent-based models) that have been applied in studying the complex physiological and pathological mechanisms of chronic inflammation in different airway diseases.

CONCLUSIONS

The scope of the present review is to explore the inflammatory mechanism in airway diseases and highlight the influence of aging on airways' inflammation mechanism. The main goal of this review is to encourage research collaborations between experimentalist and modelers to promote our understanding of the physiological and pathological mechanisms that control inflammation in different airway diseases.

(<em>b</em>)Background:</<em>b</em>) Advanced generation a<em>b</em>lation technologies have <em>b</em>een developed to achieve more effective pulmonary vein isolation (PVI) and minimize arrhythmia recurrence following atrial fi<em>b</em>rillation (AF) a<em>b</em>lation. (<em>b</em>)Methods:</<em>b</em>) We randomly assigned 346 patients with drug-refractory paroxysmal AF to contactforce guided RF a<em>b</em>lation (<em>CF</em>-RF a<em>b</em>lation, 115), 4-minute cryo<em>b</em>alloon a<em>b</em>lation (CRYO-4, 115), or 2-minute cryo<em>b</em>alloon a<em>b</em>lation (CRYO-2, 116). Follow-up was 12 months. The primary outcome was time to first documented recurrence of symptomatic or asymptomatic atrial tachyarrhythmia (AF, atrial flutter, or atrial tachycardia) <em>b</em>etween days 91 and 365 post a<em>b</em>lation, or a repeat a<em>b</em>lation procedure at any time. Secondary endpoints included freedom from symptomatic arrhythmia, and AF <em>b</em>urden. All patients received an implanta<em>b</em>le loop recorder. (<em>b</em>)Results:</<em>b</em>) One-year freedom from atrial tachyarrhythmia defined <em>b</em>y continuous rhythm monitoring, was 53.9%, 52.2%, and 51.7% with <em>CF</em>-RF, CRYO-4, and CRYO-2, respectively; P=0.87. One-year freedom from symptomatic atrial tachyarrhythmia defined <em>b</em>y continuous rhythm monitoring, was 79.1%, 78.2%, and 73.3% with <em>CF</em>-RF, CRYO-4, and CRYO-2, respectively; P=0.26. Compared to the pre-a<em>b</em>lation monitoring period, AF <em>b</em>urden was reduced <em>b</em>y a median of 99.3% (IQR 67.8-100.0%) with <em>CF</em>-RF, 99.9% (IQR 65.3-100.0%) with CRYO4, and 98.4% (IQR 56.2-100.0%) with CRYO-2 (P=0.36). Serious adverse events occurred in 2 patients in <em>CF</em>-RF (2.6%), 6 patients in CRYO-4 (5.3%), and 7 patients in CRYO-2 (6.0%), with no significant difference <em>b</em>etween groups (P=0.24). The <em>CF</em>-RF group had a significantly longer procedure duration <em>b</em>ut significantly shorter fluoroscopy exposure (P<0.001 vs. cryo<em>b</em>alloon groups). (<em>b</em>)Conclusions:</<em>b</em>) In this multicenter, randomized, single-<em>b</em>linded trial, contact-force RF a<em>b</em>lation and two different regiments of cryo<em>b</em>alloon a<em>b</em>lation resulted in no difference in one-year efficacy, which was 53% <em>b</em>y time to first recurrence <em>b</em>ut >98% <em>b</em>urden reduction as assessed <em>b</em>y continuous cardiac rhythm monitoring.

OBJECTIVE

Because allele dropout (ADO) is frequently observed in single-cell polymerase chain reaction analysis, it is important to develop a method for efficient detection of ADO, in order to avoid possible misdiagnosis in preimplantation diagnosis.

METHODS

We introduced a simultaneous amplification of mutant genes and linked polymorphic markers, such as a 4-bp repeat (GATT) at the 3' end of intron 6 in the cystic fibrosis (CF) gene and a short tandem repeat at the 5' end of the beta-globin gene. Three types of single heterozygous cells were studied for the amplification of both alleles, including 150 blastomeres, 1615 fibroblasts, and 170 first polar bodies, obtained from patients at risk for having children with cystic fibrosis (delta F-508 mutation) or sickle cell disease.

RESULTS

ADO rates of as high as 33.3% for delta F-508 mutation and 22.8% for beta-globin gene were observed in single blastomeres, compared to 7.1 and 7.7% in single fibroblasts and 5.9 and 9.6% in first polar bodies, respectively. The application of simultaneous amplification of the above linked polymorphic markers allowed detection of more than half of the cases of ADO in blastomeres (19.4% for cystic fibrosis and 12.3% for beta-globin gene) and almost all ADOs in polar bodies, particularly when the two-step sequential analysis of the first and second polar body was applied in preimplantation diagnosis of single gene disorders.

CONCLUSIONS

Simultaneous amplification of linked polymorphic markers in single-cell DNA analysis of single-gene defects is an efficient method for avoiding the risk of misdiagnosis in preimplantation diagnosis.

Persons with cystic fibrosis (CF) are susceptible to chronic pulmonary infection due to certain Burkholderia species, but it is not clear whether this typically involves persistent infection with the same strain or sequential infection with distinct strains. We analyzed 1095 Burkholderia isolates recovered from serial sputum cultures from 379 patients with CF receiving care in 112 CF treatment centers in the United States. Genotyping was performed by random amplified polymorphic DNA typing or pulsed-field gel electrophoresis. Overall, a change in infecting strain was found in 24 (6.9%) of 347 patients infected with Burkholderia cepacia complex and in 3 (9%) of 32 patients infected with Burkholderia gladioli. Several patients were likely coinfected, at least transiently, with >1 B. cepacia complex strain. The potential for strain replacement during chronic infection may confound studies of the relationship between strain and clinical outcome and must be considered in designing effective infection-control practices.

Burkholderia cepacia, a species found infrequently in cystic fibrosis (CF), was isolated from 85% of patients infected with bacteria of the B. cepacia complex that visited the major Portuguese CF center, in Lisbon, during 2003 to 2005. A detailed molecular analysis revealed that this was mainly due to two B. cepacia clones. These clones were indistinguishable from two strains isolated from intrinsically contaminated nonsterile saline solutions for nasal application, detected during routine market surveillance by the Portuguese Medicines and Health Products Authority.

Prosthetic heart valve thrombosis (PVT) is a rare but potentially life-threatening complication of heart valve replacement. An effective, quick, and easy diagnostic method is highly desirable. We evaluated the diagnostic efficacy of cine-fluoroscopy (CF), transthoracic (TTE), and transesophageal (TEE) echocardiography in 82 consecutive patients with mechanical valves and suspected PVT. Criteria for PVT were: leaflet(s) motion restriction at CF, increased Doppler gradients at TTE, and evidence of thrombi at TEE. Patients were divided in 4 groups (A, B, C, and D) according to results of CF and TTE. Group A was composed of 24 patients with positive CF and TTE. Thrombi were detected by TEE in all cases, suggesting that when both are positive, CF and TTE correctly identified PVT in all patients so that TEE may be deferred. Group B was composed of 12 patients with positive CF and negative TTE; TEE showed PVT in 4 patients (33%). These patients had very slight leaflet motion restriction as in the case of initial PVT. This suggests that CF compared with Doppler may identify patients with "hemodynamically significant" PVT. The remaining 8 patients in this group had monocuspid prostheses with negative TEE, suggesting that abnormal leaflet motion at CF may be due to functional changes. Therefore, TEE should always be performed in case of monocuspid prostheses with isolated CF abnormalities. Group C was composed of 18 asymptomatic patients with small-sized aortic prostheses and very high Doppler gradients on routine TTE. CF showed normal leaflet motion and TEE ruled out PVT in all cases outlining the diagnostic role of CF in this particular subset. Finally, group D was composed of 28 patients with negative CF and TTE. TEE did not show thrombi in 24 of 28 patients (86%), confirming that, when both yield negative results, CF and TTE are reliable methods to rule out valve thrombosis in most cases. However, in 4 of 28 patients (14%) TEE showed "nonobstructive" prosthetic thrombosis: these patients had mitral prostheses, chronic atrial fibrillation, and 3 of 4 had systemic embolisms. Thus, TEE should be performed in selected patients despite negative CF and TTE results. Sensitivity, specificity, and positive and negative predictive values were 87%, 78%, 80%, and 91% for CF and 75%, 64%, 57%, and 78% for TTE, respectively. CF and TTE correctly identified PVT in 70 of 82 patients (85%). TEE was actually required in 15% of the cases. Thus, CF and TTE are quick, effective, and complementary diagnostic tools to diagnose PVT in most patients. TEE still remains the gold standard technique in selected cases.

A phase II safety and immunogenicity study of an oral-formalin inactivated enterotoxigenic Escherichia coli (ETEC) vaccine containing six colonization factors (<em>CF</em>A/I, CS1, CS2, CS3, CS4, CS5) and 1mg of recombinant cholera toxin <em>B</em> subunit (the <em>CF</em>-<em>B</em>S-ETEC vaccine) was carried out in an urban slum of Dhaka city in <em>B</em>angladesh. The study was carried out in a double blinded, placebo controlled design in 158 children, 18-36 months of age. Children were given two doses of the <em>CF</em>-<em>B</em>S-ETEC vaccine or the placebo which consisted of E. coli K12. The vaccine was well tolerated. The immune response was studied in 60 children (30 each in the placebo and vaccine group). Significant vaccine specific IgA antibody-secreting cell (ASC) responses were seen 7 days after ingestion of the first and second dose of the vaccine. The responses to <em>CF</em>A/I (P<or=0.001), CS2 (P=0.021), CS4 (P=0.009) and rCT<em>B</em> (P<or=0.001) were elevated in the vaccines in comparison to the pre-immune values and in comparison to those seen in the placebo recipients (P=0.018 to <0.001). Vaccines but not placebo recipients also showed significantly increased IgM ASC responses to all three <em>CF</em> antigens that were tested (P=0.012 to <0.001) and IgG-ASCs to rCT<em>B</em> (P<0.001). Peak ASC levels were reached after one dose of the vaccine with no further increase or decrease after the second dose. The vaccine recipients also responded with IgA plasma antibodies to <em>CF</em>A/I, CS1, CS2, CS4 and rCT<em>B</em> after one or two doses of the vaccine (P=0.01 to <0.001). Subjects in the placebo group failed to mount responses to any of the antigens. The vaccine also induced responses in mucosal IgA antibodies in feces to <em>CF</em>A/I, CS2 and rCT<em>B</em> (61, 88 and 69% responder frequency, respectively) and the magnitude of the response was elevated in comparison to the pre-immune levels (P=0.031 to <0.001) and to the levels of the control group (P=0.003 to <0.001). This study thus shows that the <em>CF</em>-<em>B</em>S-ETEC vaccine is well tolerated in children, 18-36 months of age and gives rise to significant systemic and mucosal IgA antibody responses.

Submucosal glands (SMGs) are the major site of expression of the cystic fibrosis (CF) transmembrane conductance regulator gene (CFTR) in the human lung. As such, SMGs may be a critical component of CF lung disease pathogenesis and an important target for gene therapy. Gene-targeted mouse models exist for CF and these are used to validate gene therapy or other interventions and to dissect CF phenotypes. It is important, therefore, to compare human and mouse SMGs. We show that SMGs in the mouse are similar in structure, cell types, and Cftr expression to those in the human. Murine SMGs were found to be present in the proximal regions of the trachea at the same density as in humans but, unlike in humans, did not extend below the trachea. Upon investigation of homozygous Cftr tm1HGU and Cftr tm1G551D mutant mice, SMGs were found to extend more distally than those in wild-type control mice (P < 0.05). To investigate the development of SMGs we generated aggregation chimeric mice. Chimeric offspring contained a contribution of transgenic cells that were detectable either by DNA in situ hybridization (reiterated beta-globin transgene TgN[Hbb-bl]83Clo) or beta-galactosidase histochemistry (Lac Z reporter gene TgR[ROSA26]- 26Sor). Analysis of the distribution of transgenic cells in chimeric SMGs suggests that SMGs are clonally derived.

OBJECTIVE

Angiotensin II (Ang II) stimulates cardiac remodelling and fibrosis in the mechanically overloaded myocardium. Although Rho GTPases regulate several cellular processes, including myocardial remodelling, involvement in mediating mechanical stretch-induced regulation of angiotensinogen (Ao), the precursor to Ang II, remains to be determined. We, therefore, examined the role and associated signalling mechanisms of Rho GTPases (Rac1 and RhoA) in regulation of Ao gene expression in a stretch model of neonatal rat cardiac fibroblasts (CFs).

RESULTS

CFs were plated on deformable stretch membranes. Equiaxial mechanical stretch caused significant activation of both Rac1 and RhoA within 2-5 min. Rac1 activity returned to control levels after 4 h, whereas RhoA remained at a high level of activity until the end of the stretch period (24 h). Mechanical stretch initially caused a moderate decrease in Ao gene expression, but was significantly increased at 8-24 h. RhoA had a major role in mediating both the stretch-induced inhibition of Ao at 4 h and the subsequent upregulation of Ao expression at 24 h. β₁ integrin receptor blockade by Tac β₁ expression impaired acute (2 and 15 min) stretch-induced Rac1 activation, but increased RhoA activity. Molecular experiments revealed that Ao gene expression was inhibited by Rac1 through both JNK-dependent and independent mechanisms, and stimulated by RhoA through a p38-dependent mechanism.

CONCLUSIONS

These results indicate that stretch-induced activation of Rac1 and RhoA differentially regulates Ao gene expression by modulating p38 and JNK activation.

β-Amyloid (Aβ) peptide is the major component of senile plaques and is considered to have a causal role in the development and progression of Alzheimer’s disease (AD). There is compelling evidence supporting the notion that Aβ-induced cytotoxicity is mediated though the generation of ROS. In the present study, we investigated the neuroprotective effects of ursolic acid (UA), p-coumaric acid (p-CA), and gallic acid (GA) isolated from Corni fructus (CF) against Aβ(25-35)-induced toxicity in PC12 cell. Exposure of PC12 cells to 50 μM Aβ(25-35) increased cellular oxidative stress, the number of apoptotic cells and caspase-3 activity and finally caused significant cell death. However, UA, p-CA, and GA not only suppressed the generation of ROS but also attenuated DNA fragmentation and eventually attenuated Aβ-induced apoptosis in a dose-dependent manner. In protecting cells against Aβ neurotoxicity, UA and GA possessed stronger ability against ROS generation than p-CA, while p-CA showed the strongest anti-apoptotic activity. Particularly, p-CA protected cells at the concentration range from 0.5 up to 125 μM without any adverse effect. Taken together, these effects of UA, p-CA, and GA may be partly associated with the neuroprotective effect of CF. Furthermore, our findings might raise a possibility of therapeutic applications of CF for preventing and/or treating neurodegenerative diseases.

The cell wall of Bacillus subtilis is capable of binding different kinds of metal ions. The wall-ion complex appears to be dependent on both phosphoryl from teichoic acid and carboxylate from peptidoglycan. In the present study, cationized ferritin (CF) was used as a probe for charge distribution on the wall of B. subtilis 168. Detergent-extracted cell walls bound CF only on the outer wall face. Completed cell poles bound CF, but septa did not. When the walls were permitted to autolyze briefly, binding of CF occurred on both faces. In contrast, limited hydrolysis of the walls by egg white lysozyme resulted in the penetration of CF into the wall matrix. When walls were made teichoic acid-free, CF-binding asymmetry was preserved, suggesting that carboxyl groups were oriented toward the surface. Walls with carboxylates chemically neutralized also retained charge asymmetry. Phosphate-free and carboxyl-modified walls bound CF only poorly or not at all. These results indicate that negative charges contributed by both phosphate and carboxyl are responsible for the binding of CF and that the observed asymmetry in the distribution of the label is due to the orientation of teichoic acid and muramyl peptides toward the outside of the cell wall, above the plane of the glycan strands.

Pulmonary infections caused by Burkholderia cepacia are an important cause of morbidity and mortality in cystic fibrosis (CF) patients. Several features suggestive of invasion and intracellular sequestration of B. cepacia in CF are persistence of infection in the face of antibiotic therapy and a propensity to cause bacteraemic infections in patients with CF. A mouse respiratory challenge model was used to investigate the invasion phenotype of B. cepacia in vivo. After intratracheal inoculation, epidemic B. cepacia strains translocated from lung to liver and spleen; however, all bacteria were cleared from all organs within 7 days. B. cepacia strains, irrespective of cable piliation, were capable of attaching to and then invading murine respiratory tract epithelial cells. Histopathological examination of lungs showed interstitial infiltrates comprised mainly of polymorphonuclear leucocytes and were associated with widened alveolar septa. Electron microscopy demonstrated B. cepacia within epithelial cells and pulmonary macrophages. This study provides support for in-vitro observations that B. cepacia strains from patients with CF adhere to and then invade respiratory epithelial cells. The invasion phenotype in B. cepacia may be an important virulence factor in CF infections.

The zinc-dependent metallo-beta-lactamases are a group of bacterial enzymes that pose a threat to the future efficacy of present-day antibiotics. Their mechanism is poorly understood, and there are no clinically useful inhibitors. While most members of the group contain two tightly bound zinc ions in their active sites, the Bacillus cereus enzyme has a much lower affinity for its second zinc (Zn2), thought to be due to the presence of Arg121 immediately beneath the floor of the active site (cf. Cys/Ser/His121 in the bizinc enzymes). Crystal structures of the Arg121Cys mutant of the B. cereus 569/H/9 enzyme were solved at pH 7.0, 5.0, and 4.5, each in the presence of either 20 microM or 20 mM Zn(2+) to generate the mono- and bizinc forms, respectively. Surprisingly, the structure of the active site was unaffected by the mutation; a network of ordered water molecules replaced the interactions made by the arginine side chain, and the occupancy of Zn2 appeared minimally changed. As the pH was lowered, Zn2 moved away from one of its ligands, Asp120, but was "tracked" by two others, Cys221 and His263. Furthermore, the hydroxide ion (and proposed nucleophile for beta-lactam hydrolysis) was bound to Zn1 at pH 5 and above but absent at pH 4.5. This provides experimental evidence for an earlier proposed mechanism in which protonation of Asp120 and the Zn1-bound hydroxide are the two events that lead to the loss of activity at low pH.

BACKGROUND

Few studies have suggested the potential role of respiratory viruses in cystic fibrosis (CF) exacerbation, but their real impact is probably underestimated.

METHODS

Sixty-four sputum samples collected from 46 adult patients were included in the study: 33 samples were collected during exacerbation of CF, and 31 during the stable phase. After extraction, nucleic acids were tested for the presence of respiratory viruses. When rhinovirus (HRV) was detected, the 5'UTR and VP4/2 regions were sequenced, and phylogenetically analyzed. The characteristics of patients in exacerbation and stable phase were compared.

RESULTS

Viruses were found in 25% of samples. The HRV viruses were the most frequently detected followed by coronaviruses. Only the HRV detection was significantly associated with the occurrence of CF pulmonary exacerbation (p<0.027). Characterization of 5'UTR and VP4/2 regions of the HRV genome specified that HRV-A, -B, -C were detected. All HRV-C were recombinant HRV-Ca.

CONCLUSIONS

HRV were the most frequently detected viruses; their detection was significantly associated with the occurrence of an exacerbation. The reality of viral recombination between HRV was demonstrated in CF patients for the first time, raising the role of viruses in lung microbiota. Further studies are now warranted to decipher virus impact in CF.

The activation of dioxygen by dopamine beta-monooxygenase (DbetaM) and peptidylglycine alpha-hydroxylating monooxygenase (PHM) is postulated to occur at a copper site ligated by two histidine imidazoles and a methionine thioether, which is unusual because such thioether ligation is not present in other O2-activating copper proteins. To assess the possible role of the thioether ligand in O2 activation by DbetaM and PHM, two new ligands comprising beta-diketiminates with thioether substituents were synthesized and Cu(I) and Cu(II) complexes were isolated. The Cu(II) compounds are monomeric and exhibit intramolecular thioether coordination. While the Cu(I) complexes exhibit a multinuclear topology in the solid state, variable-temperature 1H NMR studies implicate equilibria in solution, possibly including monomers with intramolecular thioether coordination that are structurally defined by DFT calculations. Low-temperature oxygenation of solutions of the Cu(I) complexes generates stable 1:1 Cu/O2 adducts, which on the basis of combined experimental and theoretical studies adopt side-on "eta(2)" structures with negligible Cu-thioether bonding and significant peroxo-Cu(III) character. In contrast to previously reported findings with related ligands lacking the thioether group, however (cf., Aboelella; et al. J. Am. Chem. Soc. 2004, 126, 16896), purging the solutions of the thioether-containing adducts with argon results in conversion to bis(mu-oxo)dicopper(III) species. A role for the thioether in promoting loss of O2 from the 1:1 Cu/O2 adduct and facilitating trapping of the resulting Cu(I) complex to yield the bis(mu-oxo) species is proposed, and the possible relevance of this role to that of the methionine in the active sites of DbetaM and PHM is discussed.

We have characterized the subunit composition of the chloroplast ATP synthase from Chlamydomonas reinhardtii by means of a comparison of the polypeptide deficiencies in a mutant defective in photophosphorylation, with the polypeptide content in purified coupling factor (<em>CF</em>)1 and <em>CF</em>1.<em>CF</em>0 complexes. We could distinguish nine subunits in the enzyme, four of which were <em>CF</em>0 subunits. Further characterization of these subunits was undertaken by immunoblotting experiments, [14C]dicyclohexylcarbodiimide binding and analysis of their site of translation. In particular, we were able to show the presence of an as yet unidentified delta subunit in <em>CF</em>1 from C. reinhardtii. We have identified a 70-kDa peripheral membrane protein in the thylakoid membranes of C. reinhardtii, which is immunologically related to the <em>beta</em> subunit of <em>CF</em>1. We discuss its conceivable ATPase function with respect to the Ca2+-dependent ATPase activity previously reported in the thylakoid membranes from C. reinhardtii.

Based on the assumption that an accelerated proliferation process prevails in tumour cell residues after surgery, the possibility that treatment acceleration would offer a therapeutic advantage in postoperative radiotherapy of locally advanced head and neck cancer was investigated. The value of T(pot) in predicting the treatment outcome and in selecting patients for accelerated fractionation was tested. Seventy patients with (T2/N1-N2) or (T3-4/any N) squamous cell carcinoma of the oral cavity, larynx and hypopharynx who underwent radical surgery, were randomized to either (a) accelerated hyperfractionation: 46.2 Gy per 12 days, 1.4 Gy per fraction, three fractions per day with 6 h interfraction interval, treating 6 days per week or (b) Conventional fractionation: 60 Gy per 6 weeks, 2 Gy per fraction, treating 5 days per week. The 3-year locoregional control rate was significantly better in the accelerated hyperfractionation (88 +/- 4%) than in the CF (57+/- 9%) group, P=0.01 (and this was confirmed by multivariate analysis), but the difference in survival (60 +/- 10% vs 46 +/- 9%) was not significant (P=0.29). The favourable influence of a short treatment time was further substantiated by demonstrating the importance of the gap between surgery and radiotherapy and the overall treatment time between surgery and end of radiotherapy. Early mucositis progressed more rapidly and was more severe in the accelerated hyperfractionation group; reflecting a faster rate of dose accumulation. Xerostomia was experienced by all patients with a tendency to be more severe after accelerated hyperfractionation. Fibrosis and oedema also tended to be more frequent after accelerated hyperfractionation and probably represent consequential reactions. T(pot) showed a correlation with disease-free survival in a univariate analysis but did not prove to be an independent factor. Moreover, the use of the minimum and corrected P-values did not identify a significant cut-off. Compared to conventional fractionation, accelerated hyperfractionation did not seem to offer a survival advantage in fast tumours though a better local control rate was noted. This limits the use of T(pot) as a guide for selecting patients for accelerated hyperfractionation. For slowly growing tumours, tumour control and survival probabilities were not significantly different in the conventional fractionation and accelerated hyperfractionation groups. A rapid tumour growth was associated with a higher risk of distant metastases (P=0.01). In conclusion, tumour cell repopulation seems to be an important determinant of postoperative radiotherapy of locally advanced head and neck cancer despite lack of a definite association between T(pot) and treatment outcome. In fast growing tumours accelerated hyperfractionation provided an improved local control but without a survival advantage. To gain a full benefit from treatment acceleration, the surgery-radiotherapy gap and the overall treatment time should not exceed 6 and 10 weeks respectively.

Acute exacerbations of Pseudomonas aeruginosa lung infections were treated with ceftazidime (CTZ) by continuous infusion (CI) in eight adult cystic fibrosis (CF) patients. CTZ pharmacokinetics were studied after a single dose and during CI (100 mg/kg/24 h) with a CADD-PLUS infusion pump at home for 3 weeks. Individual CTZ pharmacokinetic parameters after single-dose administration were a half-life (t1/2 beta) of 1.90 +/- 0.85 h (mean +/- SD), a volume of distribution (Vs) of 0.28 +/- 0.08 L/kg, and a total body clearance (CL) of 0.152 +/- 0.014 L/h/kg. CL during CI was 0.147 +/- 0.019 L/h/kg, equal to the CL after a single dose. CTZ clearance at the start and at the end of the treatment did not differ. The mean fraction of the dose recovered from the urine was 92.6% (range 85.6-98.5%). Renal clearance was 0.147 +/- 0.015 L/h/kg and was not influenced by the pulmonary exacerbation. The early-morning serum concentrations were significantly higher than the afternoon levels (p < 0.02). The mean CTZ serum concentration during CI was 28.7 +/- 5.0 mg/L. Clinical condition and quality of life improved significantly during and after treatment. With the pharmacokinetic population data from the single-dose study, the CTZ steady-state concentrations during CI could be predicted [precision (root mean squared prediction error) 3.1 mg/L; bias (mean prediction error) 0.4 mg/L]. This method may serve as a model in the development of CTZ continuous-infusion dosage regimens in CF home treatment.

BACKGROUND

Chronic fatigue syndrome (CFS) is a disease of unknown aetiology. A patient with CFS had unexpected, marked recovery of CFS symptoms lasting for five months during and after cytotoxic chemotherapy for Hodgkin's disease. We reasoned that the transient CFS recovery was related to methotrexate treatment, which induces immunomodulation in part through B-cell depletion.

METHODS

In a case series, this patient and two additional CFS patients were B-cell depleted by infusion of the monoclonal anti-CD20 antibody rituximab.

RESULTS

All three had improvement of all CFS symptoms. Patients 1 and 2 had major amelioration from 6 weeks after intervention, patient 3 slight improvement from the same time, but then improved markedly from 26 weeks after intervention. The symptomatic effect lasted until weeks 16, 18 and 44, respectively. At relapse, all were retreated with a single (patient 1) or double rituximab infusion (patients 2 and 3). Again, all three had marked symptom improvement, mimicking their first response. After new symptom recurrence, patients 1 and 2 were given weekly oral methotrexate, patient 1 having effect also from this agent. Patients 1 and 2 were again treated for a third rituximab infusion after new relapse, again with a marked clinical benefit. No unexpected toxicity was seen.

CONCLUSIONS

These observations suggest that B-lymphocytes are involved in CFS pathogenesis for a subset of patients. Benefit for all CFS symptoms, the delayed symptom relief following B-cell depletion, the kinetics of relapses, and the effect also from methotrexate treatment, provide suggestive evidence that B-cells play a significant role in the ongoing clinical features, and that CFS may be amenable to therapeutic interventions aimed at modifying B-cell number and function. More systematic investigations of this therapeutic strategy, and of its biological basis, are now needed.

Recent molecular phylogenetic studies indicate that the rafting Indian plate harboured several isolated vertebrate lineages between ca. 130 and 56 Myr ago that dispersed and diversified 'out of India' following accretion with Eurasia. A single family of the amphibian order Gymnophiona, the Ichthyophiidae, presently occurs on the Indian plate and across much of South East Asia. Ichthyophiid phylogeny is investigated in order to test competing out of India and out of South East Asia hypotheses for their distribution. Partial sequences of mitochondrial 12S and 16S rRNA and cytochrome b genes for 20 ichthyophiids and proximate outgroups were assembled. Parsimony, maximum-likelihood and distance analyses all recover optimum trees in which uraeotyphlids plus Ichthyophis cf. malabarensis are the sister taxa to all other Ichthyophis, among which the South East Asian taxa are monophyletic. Tree topology and branch lengths indicate that the Indian lineages are more basal and older, and thus are more consistent with the hypothesis that ichthyophiids dispersed from the Indian subcontinent into South East Asia. The estimated relationships also support monophyly of Sri Lankan Ichthyophis, and non-monophyly of striped and unstriped Ichthyophis species groups. Mitochondrial DNA sequences provide evidence that should assist current problematic areas of caecilian taxonomy.