Cytochrome P450 inhibitory action of Echinacea preparations differs widely and co-varies with alkylamide content

Echinacea preparations are one of the best selling herbal medicinal products with a well established therapeutic use in the prophylaxis of upper respiratory tract infections. Their consumption is increasing, but information about their ability to inhibit cytochrome P450 enzymes (CYP) is fragmentary. The picture is further complicated by a lack of phytochemical characterization of previously tested preparations. Due to its well characterized immunomodulatory activity, the standardized Swiss registered Echinacea purpurea (L.) Moench Echinaforce extract was selected for detailed study. With the single baculovirus-expressed CYP isoforms 1A2, 2C19, 2D9 and 3A4, inhibitory actions were measured by monitoring fluorescent metabolites derived from enzyme substrates (supersome assay). The Echinaforce extract induced mild inhibition of all these isoforms, with CYP 3A4 being the most, and CYP 2D6 the least sensitive enzyme. To assess whether CYP inhibition might be a general feature of Echinacea preparations, an additional nine commercially available preparations were screened using CYP 3A4. All tested preparations were able to inhibit CYP 3A4, but inhibitory potencies (expressed as median inhibitory concentration, IC50) varied by a factor of 150. The alkylamides are thought to be responsible for the immunomodulatory activity of Echinacea, and so the concentration of 2E,4E,8Z,10E/Z-tetranoic acid isobutylamide (1) and total alkylamide content were determined in all preparations, and the latter was found to be associated with their CYP 3A4 inhibitory potency. The chemically pure alkylamides dodeca-2E,4E,8Z,10E/Z-tetranoic acid isobutylamide (1) and dodeca-2E,4E-dieonoic acid isobutylamide (2) showed inhibitory activity on CYP 2C19, 2D6 and 3A4. However, unlike the Echinaforce extract, the alkylamides did not induce CYP 1A2 inhibition. Thus, other, as yet unidentified constituents also contribute to the overall weak inhibitory effects seen with Echinacea preparations in-vitro.

Direct combination of nanoparticle fabrication and exposure to lung cell cultures in a closed setup as a method to simulate accidental nanoparticle exposure of humans

The tremendous application potential of nanosized materials stays in sharp contrast to a growing number of critical reports of their potential toxicity. Applications of in vitro methods to assess nanoparticles are severely limited through difficulties in exposing cells of the respiratory tract directly to airborne engineered nanoparticles. We present a completely new approach to expose lung cells to particles generated in situ by flame spray synthesis. Cerium oxide nanoparticles from a single run were produced and simultaneously exposed to the surface of cultured lung cells inside a glovebox. Separately collected samples were used to measure hydrodynamic particle size distribution, shape, and agglomerate morphology. Cell viability was not impaired by the conditions of the glovebox exposure. The tightness of the lung cell monolayer, the mean total lamellar body volume, and the generation of oxidative DNA damage revealed a dose-dependent cellular response to the airborne engineered nanoparticles. The direct combination of production and exposure allows studying particle toxicity in a simple and reproducible way under environmental conditions.

Virosomes can enter cells by non-phagocytic mechanisms

Phagocytosis of fine particles (1 mum) by macrophages is a ligand-receptor-mediated, actin-based process, whereas the entering of smaller particles (respiratory tract was used. We found virosomes and polystyrene particles in both populations of antigen-presenting cells, monocyte-derived macrophages, and dendritic cells, in the latter even if they were not directly exposed. In conclusion, virosomes are readily taken up by monocyte-derived macrophages, both by conventional phagocytosis and by actin-independent mechanisms. Further, they can penetrate the airway barrier and reach resident dendritic cells. Therefore, virosomes are promising vaccine candidates.

Development and pharmacokinetic characterization of pulmonal and intravenous delta-9-tetrahydrocannabinol (THC) in humans

The aim of the present study was to develop a physiologically compatible inhalation solution of delta-9-tetrahydrocannabinol (THC), and to compare the pharmacokinetic and analgesic properties of pulmonal THC versus pulmonal placebo and intravenous (iv) THC, respectively. Eight healthy volunteers were included in this randomized, double-blind, crossover study. The aqueous THC formulations were prepared by using a solubilization technique. iv THC (0.053 mg/kg body weight), pulmonal THC (0.053 mg/kg), or a placebo inhalation solution was administered as single dose. At defined time points, blood samples were collected, and somatic and psychotropic side effects as well as vital functions monitored. An ice water immersion test was performed to measure analgesia. Using a pressure-driven nebulizer, the pulmonal administration of the THC liquid aerosol resulted in high THC peak plasma levels within minutes. The bioavailability of the pulmonal THC was 28.7 +/- 8.2% (mean +/- SEM). The side effects observed after pulmonal THC were coughing and slight irritation of the upper respiratory tract, very mild psychotropic symptoms, and headache. The side effects after iv THC were much more prominent. Neither pulmonal nor iv THC significantly reduced experimentally induced pain.

Outer membrane porin M35 of Moraxella catarrhalis mediates susceptibility to aminopenicillins

BACKGROUND: The outer membrane protein M35 is a conserved porin of type 1 strains of the respiratory pathogen Moraxella catarrhalis. It was previously shown that M35 is involved in the uptake of essential nutrients required for bacterial growth and for nasal colonization in mice. The aim of this study was (i) to characterize the potential roles of M35 in the host-pathogen interactions considering the known multifunctionality of porins and (ii) to characterize the degree of conservation in the phylogenetic older subpopulation (type 2) of M. catarrhalis. RESULTS: Isogenic m35 mutants of the type 1 strains O35E, 300 and 415 were tested for their antimicrobial susceptibility against 15 different agents. Differences in the MIC (Minimum Inhibitory Concentration) between wild-type and mutant strains were found for eight antibiotics. For ampicillin and amoxicillin, we observed a statistically significant 2.5 to 2.9-fold MIC increase (p < 0.03) in the m35 mutants. Immunoblot analysis demonstrated that human saliva contains anti-M35 IgA. Wild-type strains and their respective m35 mutants were indistinguishable with respect to the phenotypes of autoagglutination, serum resistance, iron acquisition from human lactoferrin, adherence to and invasion of respiratory tract epithelial cells, and proinflammatory stimulation of human monocytes. DNA sequencing of m35 from the phylogenetic subpopulation type 2 strain 287 revealed 94.2% and 92.8% identity on the DNA and amino acid levels, respectively, in comparison with type 1 strains. CONCLUSION: The increase in MIC for ampicillin and amoxicillin, respectively, in the M35-deficient mutants indicates that this porin affects the outer membrane permeability for aminopenicillins in a clinically relevant manner. The presence of IgA antibodies in healthy human donors indicates that M35 is expressed in vivo and recognized as a mucosal antigen by the human host. However, immunoblot analysis of human saliva suggests the possibility of antigenic variation of immunoreactive epitopes, which warrants further analysis before M35 can be considered a potential vaccine candidate.

Two cases of trisomy 16 mosaicism ascertained postnatally

Postnatally ascertained trisomy 16 mosaicism is a rare diagnosis, with only three reported cases to date with no defined clinical phenotype. Trisomy 16 mosaicism diagnosed prenatally is common and associated with variable pregnancy outcomes ranging from stillbirth with multiple congenital abnormalities to an apparently normal newborn, making the genetic counseling very challenging. It is not clear whether uniparental disomy (UPD) 16 contributes to the phenotype, although it has been suggested that maternal UPD 16 affects the rate of intra-uterine growth retardation (IUGR) and congenital anomalies. We report on two further cases of trisomy 16 mosaicism confined to fibroblasts diagnosed postnatally. Patient 1 presented at birth with severe hypospadias, unilateral postaxial polydactyly, and different hair color with midline demarcation. His growth and development were normal at 11 months of age. Patient 2 was born with IUGR, significant craniofacial and body asymmetry, asymmetric skin hyperpigmentation, unilateral hearing loss, scoliosis, VSD, unexplained dilated cardiomyopathy, feeding difficulties, failure to thrive, and recurrent respiratory tract infections. She died at 7 months of age from respiratory failure. These two further cases of postnatally diagnosed trisomy 16 mosaicism highlight the variability of clinical features and outcome in this diagnosis. While Patient 2 presented with typical features of chromosomal mosaicism, Patient 1 had mild and transient features with essentially normal outcome, suggesting that trisomy 16 mosaicism may be under-diagnosed.

Cadherin-11 up-regulation in overactive bladder suburothelial myofibroblasts

PURPOSE: We investigated whether the adherens junction proteins cadherin-11 and beta-catenin can be immunohistochemically visualized in the human bladder using commercially available antibodies and, if so, whether there are differences between patients with overactive bladder and refractory detrusor overactivity, and controls without lower urinary tract symptoms. MATERIALS AND METHODS: In a prospective, nonrandomized single center study 32 patients with overactive bladder and refractory detrusor overactivity, and 8 controls without lower urinary tract symptoms underwent cystoscopic bladder biopsy. Quantitative immunohistochemistry was performed. The primary outcome was cadherin-11 and beta-catenin expression in the human bladder using commercially available antibodies. The secondary outcome was differences in cadherin-11 and beta-catenin in patients with overactive bladder and refractory detrusor overactivity, and controls. RESULTS: Double labeling experiments showed co-localization of cadherin-11 and connexin 43 in the suburothelium. There was also strong co-localization of cadherin-11 and beta-catenin in the suburothelium and detrusor. Significant 2-fold up-regulation of cadherin-11 was found in the suburothelium of patients with overactive bladder compared with that in controls (p = 0.018), whereas beta-catenin was similar in the groups (p = 0.6). In the detrusor cadherin-11 and beta-catenin expression was comparable in patients with overactive bladder and controls (each p = 0.5). No difference was observed in cadherin-11 and beta-catenin in patients with overactive bladder with idiopathic vs neurogenic detrusor overactivity in the suburothelium and the detrusor (p >0.3 and >0.2, respectively). CONCLUSIONS: Using commercially available antibodies cadherin-11 and beta-catenin expression in human bladder suburothelial myofibroblasts and detrusor smooth muscle cells was noted. Cadherin-11 up-regulation in suburothelial myofibroblasts in patients with overactive bladder may be significant in overactive bladder pathogenesis.

Clean intermittent self-catheterization after botulinum neurotoxin type A injections: short-term effect on quality of life

OBJECTIVE: To investigate the hypothesis that the need for clean intermittent self-catheterization after botulinum neurotoxin type A injections is outweighed by the efficacy of this treatment, so that clean intermittent self-catheterization is not a burden for patients with refractory idiopathic detrusor overactivity. METHODS: Women undergoing intradetrusor injections of 200 units botulinum neurotoxin type A for refractory idiopathic detrusor overactivity were evaluated prospectively. Clean intermittent self-catheterization was discussed with all patients and its possible need after botulinum neurotoxin type A treatment. As indicator of quality of life, lower urinary tract symptom distress and effect on daily activities were assessed using the validated Urogenital Distress Inventory (UDI-6) and Incontinence Impact Questionnaire (IIQ-7) before and 4 weeks after receiving botulinum neurotoxin type A injections. RESULTS: Mean age of the 65 women was 51 years, and all voided spontaneously before botulinum neurotoxin type A injections. After botulinum neurotoxin type A treatment, 28 (43%) required clean intermittent self-catheterization. Mean UDI-6 and IIQ-7 scores reduced from 61 to 33 (P<.001) and 62 to 30 (P<.001) in women performing clean intermittent self-catheterization and from 60 to 28 (P<.001) and 64 to 25 (P<.001) in those who did not, respectively. Comparison of quality of life in women performing clean intermittent self-catheterization and in those who did not revealed no significant differences before and after botulinum neurotoxin type A treatment. CONCLUSION: Clean intermittent self-catheterization after botulinum neurotoxin type A intradetrusor injections did not impair quality of life in appropriately informed and selected women in the short term. All patients should be informed of the potential need for clean intermittent self-catheterization after botulinum neurotoxin type A injections, and a willingness to do so should be a prerequisite for this still unlicensed off-label treatment.

What a patient with refractory idiopathic detrusor overactivity should know about botulinum neurotoxin type a injection

PURPOSE: We documented the effects of intradetrusor injections of botulinum neurotoxin type A (Botox(R)) for refractory idiopathic detrusor overactivity so that prospective patients maybe properly informed about possible improvement in quality of life, the duration of interinjection intervals and the risk of clean intermittent self-catheterization. MATERIALS AND METHODS: A total of 81 consecutive patients with refractory idiopathic detrusor overactivity treated with intradetrusor injections of 200 U botulinum neurotoxin type A at 20 sites per injection course were evaluated in this prospective, nonrandomized, open label cohort study. The primary outcome was changes in quality of life, as assessed by the short form of the Urogenital Distress Inventory and the Incontinence Impact Questionnaire before and after treatment. Secondary outcomes were the interinjection interval and the need for clean intermittent self-catheterization. RESULTS: After intradetrusor botulinum neurotoxin type A injections there was significant improvement in quality of life, which was sustained after repeat injections. Mean Urogenital Distress Inventory and Incontinence Impact Questionnaire scores decreased from 56 to 26 and 59 to 21 after injection 1 in 81 patients, from 52 to 30 and 51 to 24 after injection 2 in 24, from 40 to 19 and 43 to 17 after injection 3 in 13, from 44 to 17 and 61 to 15 after injection 4 in 6 and from 51 to 17 and 63 to 14 after injection 5 in 4, respectively. The median interinjection interval was 15, 12, 14 and 13 months between injections 1 and 2, 2 and 3, 3 and 4, and 4 and 5, respectively. Considering a post-void residual urine of greater than 100 ml with lower urinary tract symptoms as the indication for clean intermittent self-catheterization, the overall clean intermittent self-catheterization rate after treatment was 43%. CONCLUSIONS: Intradetrusor botulinum neurotoxin type A injections for refractory idiopathic detrusor overactivity significantly improved quality of life. This effect was sustained after repeat injection. More than 2 of 5 patients with refractory idiopathic detrusor overactivity required clean intermittent self-catheterization after botulinum neurotoxin type A injections and all prospective patients should be informed about this.

Particles induce apical plasma membrane enlargement in epithelial lung cell line depending on particle surface area dose

Background Airborne particles entering the respiratory tract may interact with the apical plasma membrane (APM) of epithelial cells and enter them. Differences in the entering mechanisms of fine (between 0.1 μm and 2.5 μm) and ultrafine ( ≤ 0.1 μm) particles may be associated with different effects on the APM. Therefore, we studied particle-induced changes in APM surface area in relation to applied and intracellular particle size, surface and number. Methods Human pulmonary epithelial cells (A549 cell line) were incubated with various concentrations of different sized fluorescent polystyrene spheres without surface charge (∅ fine – 1.062 μm, ultrafine – 0.041 μm) by submersed exposure for 24 h. APM surface area of A549 cells was estimated by design-based stereology and transmission electron microscopy. Intracellular particles were visualized and quantified by confocal laser scanning microscopy. Results Particle exposure induced an increase in APM surface area compared to negative control (p < 0.01) at the same surface area concentration of fine and ultrafine particles a finding not observed at low particle concentrations. Ultrafine particle entering was less pronounced than fine particle entering into epithelial cells, however, at the same particle surface area dose, the number of intracellular ultrafine particles was higher than that of fine particles. The number of intracellular particles showed a stronger increase for fine than for ultrafine particles at rising particle concentrations. Conclusion This study demonstrates a particle-induced enlargement of the APM surface area of a pulmonary epithelial cell line, depending on particle surface area dose. Particle uptake by epithelial cells does not seem to be responsible for this effect. We propose that direct interactions between particle surface area and cell membrane cause the enlargement of the APM.

[Antibiotic therapy in the outpatient setting: update 2009]

Rational outpatient therapy restricts antibiotics to infections where they are beneficial and selects substances based on local resistance patterns. Respiratory tract infections typically caused by viruses should not be treated with antibiotics (e.g., rhinitis, bronchitis, sinusitis). Many respiratory infections likely caused by bacteria can be treated with aminopenicillin, sometimes combined with a beta-lactamase inhibitor. Quinolones should be used only as exception for respiratory tract infections, since resistance is rising. For this reason uncomplicated urinary tract infections (cystitis) should be treated with trimethoprim-sulfa-methoxazole (TMP-SMX) instead of quinolones, even though approximately 20% of Escherichia coli are resistant to TMP-SMX. Skin and soft tissue infections are best treated with beta-lactam antibiotics, as long as the community acquired methicillin-resistant strains of S. aureus frequently seen in certain countries remain uncommon here.

Stenotrophomonas maltophilia: an emerging opportunist human pathogen

Stenotrophomonas maltophilia has emerged as an important opportunistic pathogen in the debilitated host. S maltophilia is not an inherently virulent pathogen, but its ability to colonise respiratory-tract epithelial cells and surfaces of medical devices makes it a ready coloniser of hospitalised patients. S maltophilia can cause blood-stream infections and pneumonia with considerable morbidity in immunosuppressed patients. Management of infection is hampered by high-level intrinsic resistance to many antibiotic classes and the increasing occurrence of acquired resistance to the first-line drug co-trimoxazole. Prevention of acquisition and infection depends upon the application of modern infection-control practices, with emphasis on the control of antibiotic use and environmental reservoirs.

Evolution of the leukotoxin promoter in genus Mannheimia

BACKGROUND: The Mannheimia species encompass a wide variety of bacterial lifestyles, including opportunistic pathogens and commensals of the ruminant respiratory tract, commensals of the ovine rumen, and pathogens of the ruminant integument. Here we present a scenario for the evolution of the leukotoxin promoter among representatives of the five species within genus Mannheimia. We also consider how the evolution of the leukotoxin operon fits with the evolution and maintenance of virulence. RESULTS: The alignment of the intergenic regions upstream of the leukotoxin genes showed significant sequence and positional conservation over a 225-bp stretch immediately proximal to the transcriptional start site of the lktC gene among all Mannheimia strains. However, in the course of the Mannheimia genome evolution, the acquisition of individual noncoding regions upstream of the conserved promoter region has occurred. The rate of evolution estimated branch by branch suggests that the conserved promoter may be affected to different extents by the types of natural selection that potentially operate in regulatory regions. Tandem repeats upstream of the core promoter were confined to M. haemolytica with a strong association between the sequence of the repeat units, the number of repeat units per promoter, and the phylogenetic history of this species. CONCLUSION: The mode of evolution of the intergenic regions upstream of the leukotoxin genes appears to be highly dependent on the lifestyle of the bacterium. Transition from avirulence to virulence has occurred at least once in M. haemolytica with some evolutionary success of bovine serotype A1/A6 strains. Our analysis suggests that changes in cis-regulatory systems have contributed to the derived virulence phenotype by allowing phase-variable expression of the leukotoxin protein. We propose models for how phase shifting and the associated virulence could facilitate transmission to the nasopharynx of new hosts.

RNA-Seq-based analysis of the physiologic cold shock-induced changes in Moraxella catarrhalis gene expression

BACKGROUND Moraxella catarrhalis, a major nasopharyngeal pathogen of the human respiratory tract, is exposed to rapid downshifts of environmental temperature when humans breathe cold air. The prevalence of pharyngeal colonization and respiratory tract infections caused by M. catarrhalis is greatest in winter. We investigated how M. catarrhalis uses the physiologic exposure to cold air to regulate pivotal survival systems that may contribute to M. catarrhalis virulence. RESULTS In this study we used the RNA-seq techniques to quantitatively catalogue the transcriptome of M. catarrhalis exposed to a 26 °C cold shock or to continuous growth at 37 °C. Validation of RNA-seq data using quantitative RT-PCR analysis demonstrated the RNA-seq results to be highly reliable. We observed that a 26 °C cold shock induces the expression of genes that in other bacteria have been related to virulence a strong induction was observed for genes involved in high affinity phosphate transport and iron acquisition, indicating that M. catarrhalis makes a better use of both phosphate and iron resources after exposure to cold shock. We detected the induction of genes involved in nitrogen metabolism, as well as several outer membrane proteins, including ompA, m35-like porin and multidrug efflux pump (acrAB) indicating that M. catarrhalis remodels its membrane components in response to downshift of temperature. Furthermore, we demonstrate that a 26 °C cold shock enhances the induction of genes encoding the type IV pili that are essential for natural transformation, and increases the genetic competence of M. catarrhalis, which may facilitate the rapid spread and acquisition of novel virulence-associated genes. CONCLUSION Cold shock at a physiologically relevant temperature of 26 °C induces in M. catarrhalis a complex of adaptive mechanisms that could convey novel pathogenic functions and may contribute to enhanced colonization and virulence.

The effects of thoracic epidurally administered drugs on urethral sphincter function in women: a pooled analysis

OBJECTIVES Thoracic epidural analgesia (TEA) has been shown to inhibit detrusor activity in patients undergoing open renal surgery, resulting in clinically relevant post-void residuals. However, the impact of different epidural drug mixtures on urethral sphincter function is not completely elucidated. DESIGN Pooled analysis of an open observational study and a double-blind randomized trial. SETTING Single tertiary centre. SUBJECTS Twenty-eight women without lower urinary tract symptoms and post-void residual <100 mL, who underwent open renal surgery with TEA. METHODS Pooling results in three groups with different epidural regimens (7 with bupivacaine 0.125%, 8 with bupivacaine 0.125% and fentanyl 2 μg/mL, and 13 with bupivacaine 0.1% plus fentanyl 2 μg/mL and epinephrine 2 μg/mL). All women underwent urethral pressure measurements before TEA and during TEA 2-3 days postoperatively. All patients received a TEA placed at the insertion site interspace T 8-9. RESULTS Maximum urethral closure pressure at rest decreased significantly during TEA with bupivacaine alone (median 70 cm H2 O [interquartile range 66-76] to 43 [43-65], P = 0.031) and with bupivacaine/fentanyl/epinephrine (75 cm H2 O [68-78] to 56 [52-75], P = 0.028), whereas with bupivacaine/fentanyl, no significant change could be detected (74 [51-88] vs 67 [46-70], P = 0.156). In all groups, functional profile length at rest was not influenced during TEA. CONCLUSION TEA with bupivacaine and the addition of fentanyl and epinephrine appears to decrease maximum urethral closure pressure at rest in women. The addition of fentanyl alone to bupivacaine may reduce this effect. Thus, the TEA effect on urethral sphincter function seems to depend on the drug mixture administered.