Abnormal epithelial electrolyte transport has been identified in a range of cystic fibrosis (CF) organs and appears to account for the various clinical manifestations of the disease. The aim of this study was to further define the Cl- secretion defect in CF jejunum. Excised jejunum was obtained from 11 CF patients and 12 controls. Transport studies were performed on stripped epithelium in vitro under short-circuited conditions in Ussing Chambers. 3-Isobutyl-1-methylxanthine (IBMX) (300 microM) significantly increased Cl- secretion in control (-2.3 +/- 0.6 to -3.3 +/- 0.7 mueq.cm-2.h-1; P less than 0.01, paired t test; n = 5 subjects) but not in CF jejunum (-0.5 +/- 0.3 to -0.1 +/- 0.4; n = 4). However in contrast to control jejunum, net Na+ absorption in CF jejunum was higher in the IBMX (1.3 +/- 0.5 mueq.cm-2.h-1) compared with basal periods (0.6 +/- 0.3; P less than 0.05, paired t test). IBMX stimulation of tissue adenosine 3',5'-cyclic monophosphate (cAMP) was similar in both control and CF jejunum. A range of secretagogues known to induce secretion in mammalian intestine, including dibutyryl cAMP (DBcAMP), DBcGMP, Ca2+ ionophore A23187, and the protein kinase C activator 4 beta-phorbol 12,13-dibutyrate, failed to induce secretion in CF jejunum. In conclusion, CF jejunum failed to exhibit Cl- secretion and also demonstrated abnormalities of Na+ absorption. These results support the view that the defect lies at a site distal to the intracellular messengers. Moreover, these abnormalities of intestinal electrolyte transport may account for some of the gastrointestinal manifestations of the disease such as meconium ileus and distal intestinal obstruction syndrome.

The marine sponge Diacarnus cf. spinopoculum has provided a series of norterpenes, including five new compounds (7-11), two new ent-compounds [(-)-1a and (+)-1b], and three known compounds (2a, 2b, and 12). Eight of these compounds represent additional examples of the muqubilin/sigmosceptrellin classes (norsesterterpene peroxides) or the nuapapuin class (norditerpene peroxides). Also isolated were dinorditerpenones 11 and 12, which are biosynthetically related to the muqubilin/sigmosceptrellin structure classes. In all, 11 compounds were evaluated for their cytotoxic properties using a soft agar assay system and the NCI's 60 cell-line screen. Compounds without peroxide functionality were inactive. Overall, the norsesterterpene peroxides were less selective as cytotoxins than norditerpene peroxide analogues. Two compounds, nuapapuin A methyl ester (3) and nuapapuin B (7), which were somewhat selective in their cytotoxic behavior, were selected for further in vivo evaluation.

Solute effects on the polymorphism and phase transitions in the suspensions of dipalmitoylphosphatidylcholine (DPPC) were studied by means of carboxyfluorescein (CF) and phosphatidylethanolamine rhodamine (PERho) fluorescence, differential scanning calorimetry, and X-ray diffraction. Specifically, the shifts of the lipid chain-melting phase transition, pretransition and subtransition temperature as a function of the bulk alcohol concentration were determined calorimetrically. The chain-melting phase transition temperature, Tm, was found to depend on the chain-length of the added alcohol: for short-chain alcohols (up to n-propanol), Tm first decreases and then increases with increasing alcohol concentration. For longer-chain alcohols, however, Tm decreases over the whole investigated alcohol concentration range. The pretransition and the subtransition temperature of DPPC both decrease monotonously (but non-linearly) with increasing alcohol concentration, but the former transition disappears at some characteristic, chain-length dependent alcohol concentration, cL beta i. This point in the solute-dependent phase diagram of DPPC is diagnostic of the complete hydrocarbon interdigitation. It was determined for a series of short-chain alcohols ranging from methanol through to 1-hexanol. A quantitative formula for the calculation of such limiting alcohol concentration is introduced. This formula relates the cL beta i values to the free energy of transfer of alcohols from the aqueous sub-phase into the DPPC sub-phase. By using the concept of an apparent chain-length this formalism can also be used for the alcohols with polar OH-groups at the second or third position on the hydrocarbon chain. Alcohol-induced hydrocarbon interdigitation in the phospholipid bilayers is thus shown to result chiefly from the solute-induced perturbation (lateral expansion) in the lipid headgroup region. Longer-chain alcohols, which balance this effect by disordering the phospholipid chains, therefore do not induce chain interdigitation.

OBJECTIVE

The standard dosage recommendations for beta-lactam antibiotics can result in very low drug levels in intensive care (IC) patients and burn patients in the absence of renal dysfunction. We studied the pharmacokinetic parameters and serum concentrations of ceftazidime (CF) and cefepime (CE) in burn patients and analyzed the modifications according to clinical and biological parameters and in particular age and creatinine clearance.

METHODS

Two pharmacokinetic studies were carried out with daily doses of 1 g x 6 for CF (n = 17) and 2 g x 3 for CE (n = 13). Creatinine clearance (CL(CR)) was both estimated and measured. Blood was sampled at steady state after an initial and a subsequent antibiotic dose. C(max) (maximal) and C(min) (minimal) concentrations were measured by HPLC. The influence of clinical and biological data was analyzed using ANOVA, ANCOVA and stepwise multiple linear regression.

RESULTS

The ratio of C(min) to the low MIC break point (4 mg/l) was lower than 4 in 52% of subjects receiving CF and in 80% of subjects receiving CE. The C(min) of CF was correlated with measured CL(CR) and was higher in mechanically ventilated patients than in non-ventilated patients. The clearance of CF was correlated with age. The C(min) of CE was correlated with age and drug clearance with measured CL(CR). Therefore dosage adjustment of these drugs in burn patients needs to take into account age, measured creatinine clearance and the danger of low concentrations occurring when the creatinine clearance is greater than 120 ml x min(-1).

CONCLUSIONS

In burn patients, the pharmacokinetic disposition of CF and CE was much more variable than in healthy subjects. Age and CL(CR) were predictors of the disposition of these antibiotics. Shortening the dosage interval or using continuous infusions will prevent low serum levels and keep trough levels above the MIC for longer periods of time. In view of the lack of a bedside measurement technique for ceftazidime and cefepime levels, we suggest a more frequent use of measured CL(CR) in order to attain efficacious clinical concentrations.

Post-mortem investigations of human Alzheimer's disease (AD) have largely failed to provide unequivocal evidence in support of the original amyloid cascade hypothesis, which postulated deposition of β-amyloid (Aβ) aggregates to be the cause of a demented state as well as inductive to tau neurofibrillary tangles (NFTs). Conflicting evidence suggests, however, that Aβ plaques and NFTs, albeit to a lesser extent, are present in a substantial subset of non-demented individuals. Hence, a range of soluble tau and Aβ species has more recently been implicated as the disease-relevant toxic entities. Despite the incorporation of soluble proteins into a revised amyloid cascade hypothesis, a detailed characterization of these species in the context of human AD onset, progression and cognitive decline has been lacking. Here, lateral temporal lobe samples (Brodmann area 21) of 46 human cases were profiled via tau and Aβ Western blot and native state dot blot protocols. Elevations in phospho-tau (antibodies: CP13, AT8 and PHF-1), pathological tau conformations (MC-1) and oligomeric tau (TOC1) agreed with medical diagnosis (non-AD cf. AD) and Braak stage classification (low, intermediate and high), alongside elevations in soluble Aβ species (MOAB-2 and pyro-glu Aβ) and a decline in levels of the amyloid precursor protein. Strong correlations were observed between individual Braak stages and multiple cognitive measures with all tau markers as well as total soluble Aβ. In contrast to previous reports, SDS-stable Aβ oligomers (*56) were not found to be reliable for all classifications and appeared likely to be a technical artefact. Critically, the robust predictive value of total soluble Aβ was dependent on native state quantification. Elevations in tau and Aβ within soluble fractions (Braak stage 2-3 cf. 0) were evident earlier than previously established in fibril-focused disease progression scales. Together, these data provide strong evidence that soluble forms of tau and Aβ co-localise early in AD and are closely linked to disease progression and cognitive decline.

Attempts were made to induce antibody response of hapten-primed cells by stimulation with anti-immunoglobulin (Ig) antibody and cellfree supernatant (CFS) which contained nonspecific enhancing factor. Mesenteric lymph node cells were obtained from rabbits which were primed with dinitrophenylated ragweed antigen (DNP-Rag), and the primed cells were incubated in vitro with either anti-rabbit gamma-chain or anti-Fab antibody for 24 hr. After washing, the cells were cultured in CFS which was obtained from the culture of DNP-Ascaris (DNP-Asc)-primed lymph node cells with free carrier (Asc). The results showed that hapten-primed cells were triggered for IgG anti-DNP antibody response by the two reagents, i.e., anti-Ig antibody and CFS, both which did not share any antigenic specificity with the priming antigen (DNP-Rag). Neither anti-Ig alone nor CFS alone induced anti-hapten antibody response. Anti-Ig and CFS triggered not only hapten-primed B cells but also the other IgG-bearing (B) cells for IgG synthesis. Lymph node cells of umprimed animals were activated for IgG synthesis by the stimulation with anti-Ig followed by culture in CFS. Evidence was obtained that anti-Ig antibody has to be divalent for the activation of B cells. Stimulation of DNP-primed cells with the F(ab')2 fragment of the antibody and CFS-induced anti-hapten antibody response, whereas the Fag' fragment of the anti-Ig plus CFS failed to do so. The results suggested that bridging of cell-surface Ig receptors by multivalent ligand is the initial step of B cell activation. Another evidence for the activation of hapten-primed B cells by anti-Ig was obtained by supplemental immunization of DNP-Asc-primed animals with the Fc fragment of goat IgG. Stimulation of mesenteric lymph node cells from these animals with goat anti-rabbit-Ig alone (no CFS) resulted in the formation of anti-DNP IgG antibody. The results indicated that lymphocytes primed for goat IgG collaborated with DNP-primed IgG-B cells when the lymph node cells were stimulated with goat anti-Ig.

The entomopathogenic fungi Beauveria bassiana, Lecanicillium dimorphum and L. cf. psalliotae can survive and colonize living palm tissue as endophytes. The molecular interaction between these biocontrol agent fungi and the date palm Phoenix dactylifera L. was investigated using proteomic techniques. Field date palms inoculated with these fungi were analyzed 15 and 30 days after inoculation in two independent bioassays. In vitro date palms were also inoculated with B. bassiana or L. cf. psalliotae. Qualitative and quantitative differences in protein accumulation between controls (not inoculated) and inoculated palms were found using 2-DE analysis, and some of these responsive proteins could be identified using MALDI/TOF-TOF. Proteins involved in plant defence or stress response were induced in P. dactylifera leaves as a response to endophytic colonization by entomopathogenic fungi in field date palms. Proteins related with photosynthesis and energy metabolism were also affected by entomopathogenic fungi colonization. A myosin heavy chain-like protein was accumulated in in vitro palms inoculated with these fungi. This suggests that endophytic colonization by these entomopathogenic fungi modulates plant defence responses and energy metabolism in field date palms and possibly modulates the expression of cell division-related proteins in in vitro palms at proteomic level.

The isolation of the gene responsible for the Cl- ion transport defect in cystic fibrosis (CF) has provided important information about the relationship between the disease pathology and the underlying genetic and biochemical mechanisms. In addition, new areas of investigation and therapy are now possible. Most notably, the isolation of the CF gene, the cystic fibrosis transmembrane conductance regulator (CFTR) has been led to the development of different gene therapy strategies. To circumvent possible complications due to insertional mutagenesis and virally induced immune responses, we have employed Epstein-Barr virus (EBV)-based expression vectors for correction of the cAMP-dependent Cl- transport defect associated with CF. A CFTR-containing expression construct under the regulation of the Rous sarcoma virus (RSV) long terminal repeat (LTR) (pREP5/CFTR) was introduced into transformed human airway epithelial cells defective in cAMP-dependent Cl-transport. Transfected cells were assayed for Cl- ion transport by (36)Cl- efflux, SPQ, and patch clamp and showed restoration of intact cAMP-dependent transport. CFTR transcription from pREP5/CFTR was detected by Northern hybridization. The level of response to agonists appeared to be dependent on the level of CFTR expression. When cells were tested for functional expression of CFTR after removal of selection pressure, they showed a continuous decrease in responsiveness to forskolin as a function of time after removal of selection. This decrease correlated with a loss of CFTR mRNA in the loss of the PREP5/CFTR. After 12 to 15 weeks growth without selection both cAMP-dependent Cl- transport and plasmid-derived CFTR mRNA were not detectable. However, it was still possible to rescue cAMP-dependent Cl- transport in these transfected cells by reselection suggesting the presence of the CFTR containing plasmid in a portion of the cells. Analysis of DNA indicated that the pREP5/CFTR vector copy number was reduced from 30 copies per cell with continuous selection, to approximately 0.3 copies per cell after 20 weeks without hygromycin B.

The present paper summarizes the proposed time-dependent sensitization (TDS) and partial limbic kindling model for illness from low-level chemicals; reviews and critiques prior studies on CNS aspects of multiple chemical sensitivity (MCS); and outlines possible experimental approaches to future studies. TDS is the progressive and persistent amplification of behavioral, neurochemical, endocrine, and/or immunological responses to repeated intermittent stimuli over time. Partial limbic kindling is a progressive and persistent lowering of the threshold for eliciting electrical afterdischarges, but not motor seizures, in certain brain structures such as amygdala and hippocampus; behavioral consequences include increased avoidant behaviors. The focus of the paper is the controversial claim of altered sense of smell and illness from low levels of environmental chemicals (i.e., "cacosmia"), levels that should not have any biologically harmful effects by the rules of classical neurotoxicology. A major perspective of this paper is that the phenomenology of MCS is similar to that of time-dependent sensitization (reverse tolerance) and tolerance as studied in the substance abuse literature. The TDS model for MCS proposes that neurobiological amplification underlies the symptoms and phenomenology of these patients, including their behavioral features of heightened affective and somatic distress. It is hypothesized that MCS patients, who are mostly women, may be individuals who sensitize to substances rapidly and to the extreme, to the point of aversive symptomatology with less complete capacity for development of tolerance. Possible parallels between MCS and TDS include: (a) initiation by single or multiple intermittent stimuli; (b) lasting changes in subsequent reactivity to low levels of chemically unrelated substances; (c) cross-sensitization between the stressors and pharmacological agents; (d) greater vulnerability of individuals who are female, who have certain genetic characteristics, and/or who may be hyperreactive to novelty (cf. trait shyness); (e) lack of obvious differences between sensitized and unsensitized individuals at baseline without eliciting exposures; (f) bidirectionality (bipolarity) of sensitized responses; (g) both context-dependent (conditioned) and context-independent (unconditioned) amplification of responses. To minimize variability between studies, research in this area needs (a) consensus on a working case definition of MCS or at least of cacosmia as a specific symptom in a subset of well-defined medical and psychiatric disorders; and (b) proper design of chemical challenge studies in MCS, controlling for individual differences in sensitizability and for the properties of sensitization (e.g., repeated intermittent exposure tests) and tolerance (e.g., removal from customary ambient air exposures prior to testing).

Chronic fatigue syndrome (CFS) is a heterogeneous syndrome of unknown etiology and physiopathology. CFS patients complain about disabling fatigue, depression, difficulty with memory, and concomitant skeletal and muscular pain. Interestingly enough, there is certain overlap between CFS symptoms, autoimmune rheumatic disease, and infectious diseases. Certain neuroendocrine-immune abnormalities have also been described, and autoantibodies commonly described in some autoimmune diseases have been found in CFS patients as well. An increasing number of autoantibodies, mainly directed against other nuclear cell components, have been illustrated. Likewise, an association between some infectious agents, antibody production, and later CFS onset has been reported. Similarly, vaccination is depicted as playing an important role in CFS onset. Recently, a case report pointed toward a causal association between silicone breast linkage, hepatitis B virus vaccination, and CFS onset in a previous healthy woman. Such findings suggest that there is a likely deregulation of the immune system influenced by specific agents (infections, vaccination, and products, such as silicone). Evidence suggests that CFS is a complex disease in which several risk factors might interact to cause its full expression. Thus, although different alterations have been found in CFS patients, undoubtedly the main feature is central nervous system involvement with immunological alterations. Therefore, a new term neuro-psycho-immunology must be quoted. New studies based on this concept are needed in order to investigate syndromes, such as CFS, in which immunological alterations are thought to be associated with concomitant psychological and health disturbances.

BACKGROUND

Abnormal glucose tolerance is a frequent co-morbidity in cystic fibrosis patients (CF), and is associated with a worse prognosis. The objectives are to investigate (a) the relative contribution of insulinopenia and insulin resistance (IR) for glucose tolerance and (b) the association between various glucose parameters and CF clinical status.

METHODS

Oral glucose tolerance tests were performed in 114 consecutive CF patients not known to be diabetic as well as 14 controls similar for age and BMI.

RESULTS

Abnormal glucose tolerance was found in 40% of patients with CF: 28% had impaired glucose tolerance (IGT) and 12% had new cystic fibrosis related diabetes (CFRD). Compared to control subjects, all CF patients were characterized by an increased glucose excursion (AUC). While reduced early insulin release characterised CF, IGT and CFRD patients also present IR thus both mechanisms significantly contribute to glucose tolerance abnormalities. Increased glucose AUC and reduced early insulin release but not glucose tolerance categories were associated with a reduced pulmonary function (FEV(1)).

CONCLUSIONS

In CF, early insulin secretion defect but also IR contribute to glucose intolerance. Early in the course of the disease, increased glucose AUC and reduced early insulin secretion are more closely associated with a worse clinical status than conventional glucose tolerance categories.

A wide range of data in the literature suggests that environmental enrichment has beneficial effects on various cognitive parameters in rodents. However, the magnitude of these effects and their persistence after the cessation of enrichment vary markedly across studies, with the use of different enrichment protocols probably playing a significant role in this variation. Using an open field habituation task as a paradigm, we investigate whether the duration and starting age of environmental enrichment affect the magnitude and persistence of its behavioral effects on male CF-1 albino mice. Our data shows that, at least in our protocol, (a) environmental enrichment, both after weaning and in early adulthood, decreases locomotion in an open field task, probably by increasing habituation; (b) a minimum enrichment period is necessary to induce this behavioral effect; (c) the effect of enrichment can persist at least partially for many months after its cessation; and (d) the degree of this persistence appears to be somewhat greater in animals exposed to longer durations of enrichment.

Much remains to be understood about how a group of cells or a tissue senses and regulates its size. Dictyostelium discoideum cells sense and regulate the size of groups and fruiting bodies using a secreted 450-kDa complex of proteins called counting factor (CF). Low levels of CF result in large groups, and high levels of CF result in small groups. We previously found three components of CF (D. A. Brock and R. H. Gomer, Genes Dev. 13:1960-1969, 1999; D. A. Brock, R. D. Hatton, D.-V. Giurgiutiu, B. Scott, R. Ammann, and R. H. Gomer, Development 129:3657-3668, 2002; and D. A. Brock, R. D. Hatton, D.-V. Giurgiutiu, B. Scott, W. Jang, R. Ammann, and R. H. Gomer, Eukaryot. Cell 2:788-797, 2003). We describe here a fourth component, CFCFCFCF when a different CF component, CFCFCFCFCFCFCFCF component CFCFCFCFCFCF, and thus this secreted signal has four different protein components.

We investigated the dynamics and diversity of heterotrophic bacteria, autotrophic and heterotrophic flagellates, and ciliates from March to July 2002 in the surface waters (0-50 m) of Lake Bourget. The heterotrophic bacteria consisted mainly of "small" cocci, but filaments (>2 microm), commonly considered to be grazing-resistant forms under increased nanoflagellate grazing, were also detected. These elongated cells mainly belonged to the Cytophaga-Flavobacterium (CF) cluster, and were most abundant during spring and early summer, when mixotrophic or heterotrophic flagellates were the main bacterial predators. The CF group strongly dominated fluorescent in situ hybridization-detected cells from March to June, whereas clear changes were observed in early summer when Beta-proteobacteria and Alpha-proteobacteria increased concomitantly with maximal protist grazing pressures. The analysis of protist community structure revealed that the flagellates consisted mainly of cryptomonad forms. The dynamics of Cryptomonas sp. and Dinobryon sp. suggested the potential importance of mixotrophs as consumers of bacteria. This point was verified by an experimental approach based on fluorescent microbeads to assess the potential grazing impact of all protist taxa in the epilimnion. From the results, three distinct periods in the functioning of the epilimnetic microbial loop were identified. In early spring, mixotrophic and heterotrophic flagellates constituted the main bacterivores, and were regulated by the availability of their resources mainly during April (phase 1). Once the "clear water phase" was established, the predation pressure of metazooplankton represented a strong top-down force on all microbial compartments. During this period only mixotrophic flagellates occasionally exerted a significant bacterivory pressure (phase 2). Finally, the early summer was characterized by the highest protozoan grazing impact and by a rapid shift in the carbon pathway transfer, with a fast change-over of the main predators contribution, i.e., mixotrophic, heterotrophic flagellates and ciliates in bacterial mortality. The high abundance of ciliates during this period was consistent with the high densities of resources (heterotrophic nanoflagellates, algae, bacteria) in deep layers containing the most chlorophyll. Bacteria, as ciliates, responded clearly to increasing phytoplankton abundance, and although bacterial grazing impact could vary largely, bacterial abundance seemed to be primarily bottom-up regulated (phase 3).

OBJECTIVE

To evaluate the clinical relevance of each cofactor on clinical presentation of chronic hepatitis B.

METHODS

Out of 1366 hepatitis B surface antigen (HBsAg) positive subjects consecutively observed in 79 Italian hospitals, 53 (4.3%) showed as the only cofactor hepatitis D virus (HDV) infection [hepatitis B virus (HBV)/HDV group], 130 (9.5%) hepatitis C virus (HCV) (group HBV/HCV), 6 (0.4%) human immunodeficiency virus (HIV) (group HBV/HIV), 138 (10.2%) alcohol abuse (group HBV/alcohol); 109 (8.0%) subjects had at least two cofactors and 924 were in the cofactor-free (CF) group.

RESULTS

Compared with patients in group CF those in group HBV/alcohol were older and more frequently had cirrhosis (P < 0.001), those in group HBV/HDV were younger (P < 0.001), more frequently resided in the south of the country and had cirrhosis (P <0.001), those in group HBV/HCV were older (P < 0.001) and more frequently had cirrhosis (P < 0.001). These cofactors were all independent predictors of liver cirrhosis in HBsAg positive patients. Multivariate analysis showed that an older age [odds ratio (OR) 1.06, 95% CI: 1.05-1.08], alcohol abuse with more than 8 drinks daily (OR 2.89, 95% CI: 1.81-4.62) and anti-HDV positivity (OR 3.48, 95% CI: 2.16-5.58) are all independently associated with liver cirrhosis. This association was found also for anti-HCV positivity in univariate analysis, but it was no longer associated (OR 1.23, 95% CI: 0.84-1.80) at multivariate analysis.

CONCLUSIONS

Older age, HDV infection and alcohol abuse are the major determinants of severe liver disease in chronic HBV infection, while HCV replication plays a lesser role in the severity of hepatic damage.

MicroRNA-7a/b (miR-7a/b) protects cardiac myocytes from apoptosis during ischemia/reperfusion injury; however, its role in angiotensin II (ANG II)-stimulated cardiac fibroblasts (CFs) remains unknown. Therefore, the present study investigated the anti-fibrotic mechanism of miR-7a/b in ANG II-treated CFs. ANG II stimulated the expression of specific protein 1 (Sp1) and collagen I in a dose- and time-dependent manner, and the overexpression of miR-7a/b significantly down-regulated the expression of Sp1 and collagen I stimulated by ANG II (100 nM) for 24 h. miR-7a/b overexpression effectively inhibited MMP-2 expression/activity and MMP-9 expression, as well as CF proliferation and migration. In addition, miR-7a/b also repressed the activation of TGF-β, ERK, JNK and p38 by ANG II. The inhibition of Sp1 binding activity by mithramycin prevented collagen I overproduction; however, miR-7a/b down-regulation reversed this effect. Further studies revealed that Sp1 also mediated miR-7a/b-regulated MMP expression and CF migration, as well as TGF-β and ERK activation. In conclusion, miR-7a/b has an anti-fibrotic role in ANG II-treated CFs that is mediated by Sp1 mechanism involving the TGF-β and MAPKs pathways.

OBJECTIVE

The cystic fibrosis (CF) airway is now considered the site of a complex microbiota, where cross-talking between microbes and lateral gene transfer are believed to contribute to the adaptation of bacteria to this specific environment and to the emergence of multidrug-resistant bacteria. The objective of this study was to retrieve and analyse specific sequences associated with antimicrobial resistance from the CF viromes database.

METHODS

Specific sequences from CF metagenomic studies related to the 'antibiotic and toxic compound resistance' dataset were retrieved from the MG-RAST web site, assembled and functionally annotated for identification of the genes. Phylogenetic trees were constructed using a minimum parsimony starting tree topology search strategy.

RESULTS

Overall, we found 1031 short sequences in the CF virome putatively encoding resistance to antimicrobials versus only 3 reads in the non-CF virome dataset (P = 0.001). Among them, we could confidently identify 66 efflux pump genes, 15 fluoroquinolone resistance genes and 9 β-lactamase genes. Evolutionary relatedness determined using phylogenetic information demonstrates the different origins of these genes among the CF microbiota. Interestingly, among annotated sequences within CF viromes, we also found matching 16S rDNA sequences from Escherichia, Cyanobacteria and Bacteroidetes.

CONCLUSIONS

Our results suggest that phages in the CF sputum microbiota represent a reservoir of mobilizable genes associated with antimicrobial resistance that may spread in this specific niche. This phenomenon could explain the fantastic adaptation of CF strains to their niche and may represent a new potential therapeutic target to prevent the emergence of multidrug-resistant bacteria, which are responsible for most of the deaths in CF.

OBJECTIVE

Orthostatic hypotension has a prognostic role in determining cardiovascular and all-cause mortality. The aim of this study was to assess the prevalence of orthostatic hypotension and its association with blood pressure (BP) levels, arterial stiffness, cardiovascular and metabolic disorders and medication in individuals aged 80 years and over living in nursing home.

METHODS

In 994 individuals (77% women, mean age 88±5 years), the presence of orthostatic hypotension was tested according to American Autonomic Society and American Academy of Neurology guidelines. Arterial stiffness was evaluated with carotid-femoral pulse wave velocity (cf-PWV), peripheral to central pulse pressure amplification (PPA) and augmentation index. Cardiovascular and metabolic disorders as well as medications were recorded from patients' medical records.

RESULTS

The prevalence of orthostatic hypotension was 18%. Treated hypertensive patients with SBP 140 mmHg or less had a lower prevalence of orthostatic hypotension than patients with SBP more than 140 mmHg (respectively, 13 vs. 23%; P < 0.001). Individuals with orthostatic hypotension exhibited higher brachial and central PP than individuals without orthostatic hypotension (respectively, 69±18 vs. 65±16 mmHg and 57±17 vs. 54±15 mmHg; P < 0.01). In these same individuals, a significant increase in augmentation index (31.1±14.0 vs. 27.2±13.6%; P < 0.01), but not in cf-PWV or in PPA, was observed. Individuals with orthostatic hypotension were treated more frequently with β-blockers and less frequently with angiotensin receptor blockers or nitrates than individuals without orthostatic hypotension (P < 0.05 for both).

CONCLUSIONS

Contrary to the general belief, elderly individuals with well controlled BP (SBP < 140 mmHg) show lower orthostatic hypotension, thus constituting a complementary argument for efficaciously treating hypertension in these individuals.

Sequences of the internal transcribed spacer region 1 (ITS1) of the ribosomal DNA were used to determine the phylogenetic relationships of species of Trichoderma sect. Pachybasium. To this end, 85 strains-including all the available ex-type strains-were analyzed. Parsimony analysis demonstrated that the section is nonmonophyletic, distributing the 85 strains among three main groups that were supported by bootstrap values. Group A comprises two clades (A1 and A2), with A1 including T. polysporum, T. piluliferum, and T. minutisporum, while A2 included T. hamatum, T. pubescens, and T. strigosum in addition to species previously included in sect. Trichoderma (i.e., T. viride, T. atroviride, and T. koningii). The ex-type strain of T. fasciculatum formed a separate branch basal to clade A. Clade B contained the sect. Pachybasium members T. harzianum, T. fertile, T. croceum, T. longipile, T. strictipile, T. tomentosum, T. oblongisporum, T. flavofuscum, T. spirale, and the anamorphs of Hypocrea semiorbis and H. cf. gelatinosa. Sequence differences among clades A1, A2, and B were in the same order of magnitude as between each of them and T. longibrachiatum, which was used as an outgroup in these analyses. Sequence differences within clades A1, A2, and B were considerably smaller: in some cases (i.e., T. virens and T. flavofuscum; T. strictipile and H. cf. gelatinosa), the ITS1-sequences were identical, suggesting conspecifity. In other cases (e.g., T. crassum and T. longipile; T. harzianum, T. inhamatum, T. croceum, T. fertile, and H. semiorbis; T. hamatum and T. pubescens; and T. viride, T. atroviride, and T. koningii) differences were in the range of 1-3 nt only, suggesting a very close phylogenetic relationship. The sequence of a previously described aggressive mushroom competitor group of T. harzianum strains (Th2) was strikingly different from that of the ex-type strain of T. harzianum and closely related species and is likely to be a separate species. Copyright 1998 Academic Press.

OBJECTIVE

Progression and severity of lung disease differs markedly and early between patients with cystic fibrosis (CF). We investigated the hypothesis that polymorphisms in the detoxifying enzymes glutathione-S-transferase (GST) could influence phenotypic presentation of lung disease in CF.

METHODS

Genotypes for GSTM1, GSTM3, GSTP1 and GSTT1 were determined in a cohort of 146 children with CF by PCR-based methods. Pulmonary function, assessed by spirometric measures of forced expiratory volume in one second (FEV1) and forced vital capacity (FVC), was analysed in children at the age of 9.

RESULTS

No association between spirometric measurements, and GSTM1, GSTP1 or GSTT1 genotypes was found. As compared with patients homozygous for GSTM3*A allele, CF children carrying the GSTM3*B allele displayed a significant better lung function, assessed by both mean values of FEV1 and of FVC (respectively P = 0.01 and P = 0.002). These correlations remained significant after adjustment for potential confounding factors (respectively adjusted P = 0.008 and P = 0.002) and also in subgroups of CF patients who carry the deltaF508 CFTR mutation. Haplotype analysis of GSTM3 in combination with GSTM1 indicated that the positive impact of GSTM3*B allele on pulmonary performances was barely influenced by the GSTM1 genotypes of CF children.

CONCLUSIONS

These data provide the first evidence suggesting that polymorphism of the GSTM3 gene contributes to clinical severity in CF, which may have prognostic significance and could prompt to start a more targeted therapy in young patients with CF.

OBJECTIVE

Activation of cardiac fibroblasts into myofibroblasts constitutes a key step in cardiac remodeling after myocardial infarction (MI), due to interstitial fibrosis. Mesenchymal stem cells (MSCs) have been shown to improve post-MI remodeling an effect that is enhanced by hypoxia preconditioning (HPC). Leptin has been shown to promote cardiac fibrosis. The expression of leptin is significantly increased in MSCs after HPC but it is unknown whether leptin contributes to MSC therapy or the fibrosis process. The objective of this study was to determine whether leptin secreted from MSCs modulates cardiac fibrosis.

METHODS

Cardiac fibroblast (CF) activation was induced by hypoxia (0.5% O2). The effects of MSCs on fibroblast activation were analyzed by co-culturing MSCs with CFs, and detecting the expression of α-SMA, SM22α, and collagen IαI in CFs by western blot, immunofluorescence and Sirius red staining. In vivo MSCs antifibrotic effects on left ventricular remodeling were investigated using an acute MI model involving permanent ligation of the left anterior descending coronary artery.

RESULTS

Co-cultured MSCs decreased fibroblast activation and HPC enhanced the effects. Leptin deficit MSCs from Ob/Ob mice did not decrease fibroblast activation. Consistent with this, H-MSCs significantly inhibited cardiac fibrosis after MI and mediated decreased expression of TGF-β/Smad2 and MRTF-A in CFs. These effects were again absent in leptin-deficient MSCs.

CONCLUSIONS

Our data demonstrate that activation of cardiac fibroblast was inhibited by MSCs in a manner that was leptin-dependent. The mechanism may involve blocking TGF-β/Smad2 and MRTF-A signal pathways.

Despite emerging data indicating a role for T cells in profibrotic cardiac repair and healing after ischemia, little is known about whether T cells directly impact cardiac fibroblasts (CFBs) to promote cardiac fibrosis (CF) in nonischemic heart failure (HF). Recently, we reported increased T cell infiltration in the fibrotic myocardium of nonischemic HF patients, as well as the protection from CF and HF in TCR-α-/- mice. Here, we report that T cells activated in such a context are mainly IFN-γ+, adhere to CFB, and induce their transition into myofibroblasts. Th1 effector cells selectively drive CF both in vitro and in vivo, whereas adoptive transfer of Th1 cells, opposite to activated IFN-γ-/- Th cells, partially reconstituted CF and HF in TCR-α-/- recipient mice. Mechanistically, Th1 cells use integrin α4 to adhere to and induce TGF-β in CFB in an IFN-γ-dependent manner. Our findings identify a previously unrecognized role for Th1 cells as integrators of perivascular CF and cardiac dysfunction in nonischemic HF.

BACKGROUND

Transient receptor potential melastatin 3 (TRPM3) cation channels are ubiquitously expressed by multiple cells and have an important regulatory role in calcium-dependent cell signalling to help maintain cellular homeostasis. TRPM3 protein expression has yet to be determined on Natural Killer (NK) cells and B lymphocytes. Multiple single nucleotide polymorphisms have been reported in TRPM3 genes from isolated peripheral blood mononuclear cells, NK and B cells in Chronic fatigue syndrome/Myalgic encephalomyelitis (CFS/ME) patients and have been proposed to correlate with illness presentation. The object of the study was to assess TRPM3 surface expression on NK and B lymphocytes from healthy controls, followed by a comparative investigation examining TRPM3 surface expression, and cytoplasmic and mitochondrial calcium influx in CD19(+) B cells, CD56(bright) and CD56(dim) cell populations from CFS/ME patients.

RESULTS

TRPM3 cell surface expression was identified for NK and B lymphocytes in healthy controls (CD56(bright) TRPM3 35.72 % ± 7.37; CD56(dim) 5.74 % ± 2.00; B lymphocytes 2.05 % ± 0.19, respectively). There was a significant reduction of TRPM3 surface expression on CD19(+) B cells (1.56 ± 0.191) and CD56(bright) NK cells (17.37 % ± 5.34) in CFS/ME compared with healthy controls. Anti-CD21 and anti-IgM conjugated biotin was cross-linked with streptavidin,and subsequently treatment with thapsigargin. This showed a significant reduction in cytoplasmic calcium ion concentration in CD19(+) B lymphocytes. CD56(bright) NK cells also had a significant decrease in cytoplasmic calcium in the presence of 2-APB and thapsigargin in CFS/ME patients.

CONCLUSIONS

The results from this preliminary investigation identify, for the first time, TRPM3 surface expression on both NK and B lymphocytes in healthy controls. We also report for the first time, significant reduction in TRPM3 cell surface expression in NK and B lymphocytes, as well as decreased intracellular calcium within specific conditions in CFS/ME patients. This warrants further examination of these pathways to elucidate whether TRPM3 and impaired calcium mobilisation has a role in CFS/ME.

BACKGROUND

Unremitting fatigue and unrefreshing sleep, hallmark traits of Chronic Fatigue Syndrome (CFS), are also pathognomonic of sleep disorders. Yet, no reproducible perturbations of sleep architecture, multiple sleep latency times or Epworth Sleepiness Scores are found to be associated consistently with CFS. This led us to hypothesize that sleep homeostasis, rather than sleep architecture, may be perturbed in CFS. To probe this hypothesis, we measured and compared EEG frequencies associated with restorative sleep between persons with CFS and matched controls, both derived from a population-based sample.

METHODS

We evaluated overnight polysomnography (PSG) in 35 CFS and 40 control subjects. PSG records were manually scored and epochs containing artifact removed. Fast Fourier Transformation was utilized to deconstruct individual EEG signals into primary frequency bands of alpha, delta, theta, sigma, and beta frequency domains. The spectral power of each frequency domain for each sleep state was compared between persons with CFS and matched controls.

RESULTS

In persons with CFS, delta power was diminished during slow wave sleep, but elevated during both stage 1 and REM. Alpha power was reduced during stage 2, slow wave, and REM sleep. Those with CFS also had significantly lower theta, sigma, and beta spectral power during stage 2, Slow Wave Sleep, and REM.

CONCLUSIONS

Employing quantitative EEG analysis we demonstrate reduced spectral power of cortical delta activity during SWS. We also establish reduced spectral power of cortical alpha activity, with the greatest reduction occurring during REM sleep. Reductions in theta, beta, and sigma spectral power were also apparent.

CONCLUSIONS

Unremitting fatigue and unrefreshing sleep, the waking manifestations of CFS, may be the consequence of impaired sleep homeostasis rather than a primary sleep disorder.