Wnt5a Inhibits Human Monocyte‐Derived Myeloid Dendritic Cell Generation

Wnt5a is a non‐canonical Wnt protein that is expressed at elevated levels in inflammatory conditions. Its role in inflammation remains unclear, although it is known that Wnt5a is expressed at a higher level in monocyte‐derived myeloid dendritic cells (Mo‐mDCs) than in monocytes and macrophages. The function of Wnt5a in dendritic cells (DCs) remains relatively unexplored. Here, we found that under Mo‐mDC culture conditions, Wnt5a inhibited the generation of CD14^+/low Mo‐mDCs while promoting the generation of CD14^+/++ CD16⁺ monocytes. We could further show that stimulation of monocytes with rWnt5a induced a rapid IL‐6 production and that the rWnt5a treated Mo‐mDC differentiation was restored upon blocking of IL‐6. Also, conditioned media from Wnt5a stimulated human breast cancer cells producing IL‐6, specifically inhibited Mo‐mDC differentiation. These observations are strengthened by our finding that patients with sepsis, a disease involving elevated Wnt5a and IL‐6 levels, also showed a significant increase in the CD14⁺ CD16⁺⁺/CD14^+/++ CD16⁺ monocyte populations, which was accompanied by a significant decrease in circulating mDCs. We finally show that under typical Mo‐mDC culture conditions, monocytes isolated from patients with sepsis as compared to healthy controls, preferentially differentiated into CD14^+/++ HLA‐DR⁺⁺ cells. We suggest that Wnt5a is a possible candidate mediator for the CD14^+/++ CD16⁺ monocyte accumulation seen in patients with infectious disease and cancer.     

Multiple point mutations in a shuttle vector propagated in human cells: evidence for an error-prone DNA polymerase activity.

Mutagenesis was studied at the DNA-sequence level in human fibroblast and lymphoid cells by use of a shuttle vector plasmid, pZ189, containing a suppressor tRNA marker gene. In a series of experiments, 62 plasmids were recovered that had two to six base substitutions in the 160-base-pair marker gene. Approximately 20-30% of the mutant plasmids that were recovered after passing ultraviolet-treated pZ189 through a repair-proficient human fibroblast line contained these multiple mutations. In contrast, passage of ultraviolet-treated pZ189 through an excision-repair-deficient (xeroderma pigmentosum) line yielded only 2% multiple base substitution mutants. Introducing a single-strand nick in otherwise unmodified pZ189 adjacent to the marker, followed by passage through the xeroderma pigmentosum cells, resulted in about 66% multiple base substitution mutants. The multiple mutations were found in a 160-base-pair region containing the marker gene but were rarely found in an adjacent 170-base-pair region. Passing ultraviolet-treated or nicked pZ189 through a repair-proficient human B-cell line also yielded multiple base substitution mutations in 20-33% of the mutant plasmids. An explanation for these multiple mutations is that they were generated by an error-prone polymerase while filling gaps. These mutations share many of the properties displayed by mutations in the immunoglobulin hypervariable regions.     

Neurotrophic factor intervention restores auditory function in deafened animals

A primary cause of deafness is damage of receptor cells in the inner ear. Clinically, it has been demonstrated that effective functionality can be provided by electrical stimulation of the auditory nerve, thus bypassing damaged receptor cells. However, subsequent to sensory cell loss there is a secondary degeneration of the afferent nerve fibers, resulting in reduced effectiveness of such cochlear prostheses. The effects of neurotrophic factors were tested in a guinea pig cochlear prosthesis model. After chemical deafening to mimic the clinical situation, the neurotrophic factors brain-derived neurotrophic factor and an analogue of ciliary neurotrophic factor were infused directly into the cochlea of the inner ear for 26 days by using an osmotic pump system. An electrode introduced into the cochlea was used to elicit auditory responses just as in patients implanted with cochlear prostheses. Intervention with brain-derived neurotrophic factor and the ciliary neurotrophic factor analogue not only increased the survival of auditory spiral ganglion neurons, but significantly enhanced the functional responsiveness of the auditory system as measured by using electrically evoked auditory brainstem responses. This demonstration that neurotrophin intervention enhances threshold sensitivity within the auditory system will have great clinical importance for the treatment of deaf patients with cochlear prostheses. The findings have direct implications for the enhancement of responsiveness in deafferented peripheral nerves.     

Targeting versus tinkering: Explaining why the clinic is frustrated with molecular mapping of disease mechanisms

We argue that our common diseases should not necessarily be taken as a sign of physiological error. Regulatory networks developed by evolutionary forces to support reproductive fitness happen to include disease as a side-effect. For example, inflammatory and autoimmune diseases are secondary to a strong defence against infections. An evolutionary perspective can help us understand why many drugs targeted to single molecules or linear signaling pathways fail in clinical trials. We present the hypothesis that a tinkering research strategy, as compared with the prevailing reductionist approach, may be more likely to help us find the tools needed to interfere optimally with disease-generating networks. One application of the hypothesis can be to analyze how manipulation with diet and gut microbial flora influences multiple sclerosis patients, rather than to first map in detail the molecular disease mechanism and then develop targeting drugs.     

Enhanced DNA damage-induced p53 peptide phosphorylation and cell-cycle arrest in Sjögren's syndrome cells

Background Cells from primary Sjögren’s syndrome (SS) patients have been reported to show alterations in DNA repair and p53 expression. The DNA‐dependent protein kinase (DNA‐PK) autoantigen may be involved in both of these alterations in relation to cellular DNA damage responses. We conducted this study of cell‐cycle kinetics and p53 to find additional evidence for an abnormal stress response role in the pathogenesis of SS. Design DNA‐dependent protein kinase activity, p53 peptide phosphorylation and p53 protein levels were determined in gamma‐irradiated long‐term T lymphocyte cultures. Cell‐cycle progression of peripheral blood mononuclear cells was analysed with flow cytometry. Results No significant differences in the DNA‐PK activities or p53 protein levels appeared between the SS patients and the healthy individuals. However, patients with the SS hallmark Ro/SS‐A and La/SS‐B autoantibodies showed enhancement of both p53 peptide phosphorylation (P = 0·036) and G1 cell‐cycle arrest (P = 0·015) in response to gamma radiation. Conclusions Sjögren’s syndrome cells express an enhanced G1 checkpoint function which may be mediated partly by p53 phosphorylation, suggesting that an abnormal stress response in SS is of relevance for the development of this autoimmune disease.     

Pharmacokinetics and pharmacodynamics of ximelagatran,a novel oral direct thrombin inhibitor,in young healthy male subjects

OBJECTIVES: Ximelagatran is a novel, oral direct thrombin inhibitor designed to overcome the low and variable oral absorption of melagatran, its active form. The pharmacokinetics and pharmacodynamics of ximelagatran following single and repeated oral administration were investigated. The primary objectives were to determine the dose linearity and reproducibility of melagatran exposure and the influence of food intake. METHODS: Two open-label studies were performed in healthy male subjects. Study I was a dose-escalation study, in which subjects received single oral doses of ximelagatran (1-98 mg). Study II was a randomised, two-way crossover study consisting of two 5-day treatment periods, in which subjects received a 20-mg oral dose of ximelagatran twice daily, either before breakfast and with dinner, or with breakfast and after dinner. RESULTS: Ximelagatran was rapidly absorbed and converted to melagatran, which was the predominant compound in plasma. The mean (+/- standard deviation) bioavailability of melagatran was 22.2+/-4.3% and 17.4+/-2.8% after single and repeated dosings, respectively. The maximum plasma concentration of melagatran and the area under the melagatran plasma concentration-time curve (AUC) increased linearly with dose. Inter- and intra-subject variability in melagatran AUC was 8% and 12%, respectively, with no relevant food- or time dependence. Anticoagulation, assessed as activated partial thromboplastin time, was correlated with melagatran plasma concentration. There was virtually no increase in capillary bleeding time over the dose range studied, and ximelagatran was well tolerated. CONCLUSION: After oral administration of ximelagatran to healthy male subjects, the pharmacokinetic and pharmacodynamic profile of melagatran is predictable and reproducible.     

CT diagnosis of temporal bone anomalies and cochlear implant surgery in CHARGE association

Children with CHARGE have multiple disabilities. Impairments of vision and hearing, balance problems and facial palsy are common. Few reports have dealt with their radiological temporal bone changes and none with cochlear implant surgery. The pre-operative temporal bone computed tomography (CT) and surgical findings of one child with CHARGE and one with a CHARGE-like condition who have received cochlear implants are reported. The findings include absent semicircular canals, narrow orifice for the cochlear nerve, and abnormalities of the oval and round window, the facial canal and the ossicles. CT can be used as a diagnostic tool as these combinations of temporal bone changes are extremely rare in other materials. Cochlear implants may help these often very isolated children to communicate.     

Pharmacokinetics of esomeprazole after oral and intravenous administration of single and repeated doses to healthy subjects

OBJECTIVE: To study the pharmacokinetics of esomeprazole, one of the optical isomers of omeprazole, after 20 mg or 40 mg single and repeated oral and intravenous administration to healthy subjects. The main metabolites of esomeprazole were also assessed after the 40-mg oral dose. METHODS: In two separate studies, 16 healthy male subjects and 16 healthy male and female subjects received intravenous doses of 20 mg and 40 mg esomeprazole, respectively, on the first investigation day. After a washout period of 5-14 days, the same doses (20 mg as a solution and 40 mg as a capsule) were given orally for 5 days and then again intravenously on day 6. Blood samples for determination of esomeprazole and its metabolites were collected 12 h or 24 h post-dose and were analysed using normal-phase liquid chromatography with ultraviolet (UV) detection. Pharmacokinetic parameters of esomeprazole and its metabolites were estimated using non-compartmental analysis. Geometric means and ratios of the geometric means together with 95% confidence intervals (CI) of the pharmacokinetic parameters were calculated using analysis of variance (ANOVA). RESULTS: Plasma clearance (CL) of esomeprazole decreased from 22 l/h to 16 l/h and from 17 l/h to 9 l/h following repeated dosing of 20 mg and 40 mg, respectively. Total area under the plasma concentration-time curve (AUC) increased (from 1.34 micromol x h/l to 2.55 micromol x h/l) with absolute bioavailability (F) being 50% on day 1 and 68% on day 5 after the 20-mg oral dose. AUC increased (from 4.32 micromol x h/l to 11.21 micromol x h/l) with F being 64% on day 1 and 89% on day 5 after the 40-mg oral dose. The plasma levels for esomeprazole sulphone were substantially higher on day 5 than on day 1, while those for 5-hydroxy esomeprazole were marginally higher on day 5 than on day 1 following repeated oral dosing of 40 mg esomeprazole. No side effects attributable to esomeprazole were noticed. CONCLUSION: The increased AUC of esomeprazole with repeated dosing is probably due to a combination of a decreased first-pass elimination and a decreased systemic clearance.     

Improved measurement of drug exposure in the brain using drug-specific correction for residual blood

A major challenge associated with the determination of the unbound brain-to-plasma concentration ratio of a drug (K_p,uu,brain), is the error associated with correction for the drug in various vascular spaces of the brain, i.e., in residual blood. The apparent brain vascular spaces of plasma water (V_water, 10.3 μL/g brain), plasma proteins (V_protein, 7.99 μL/g brain), and the volume of erythrocytes (V_er, 2.13 μL/g brain) were determined and incorporated into a novel, drug-specific correction model that took the drug-unbound fraction in the plasma (f_u,p) into account. The correction model was successfully applied for the determination of K_p,uu,brain for indomethacin, loperamide, and moxalactam, which had potential problems associated with correction. The influence on correction of the drug associated with erythrocytes was shown to be minimal. Therefore, it is proposed that correction for residual blood can be performed using an effective plasma space in the brain (V_eff), which is calculated from the measured f_u,p of the particular drug as well as from the estimates of V_water and V_protein, which are provided in this study. Furthermore, the results highlight the value of determining K_p,uu,brain with statistical precision to enable appropriate interpretation of brain exposure for drugs that appear to be restricted to the brain vascular spaces.