Simultaneous targeting of HCV replication and viral binding with a single lentiviral vector containing multiple RNA interference expression cassettes.

Chronic hepatitis C virus (HCV) infection has a major medical impact and current treatments are often unsuccessful. RNA interference represents a promising new approach to tackling this problem. The current study details the design and testing of self-inactivating lentiviral vectors (LV) delivering RNA interference to prevent HCV replication and infection. Vectors were constructed with single, double, and triple cassettes expressing short hairpin RNAs (shRNAs) simultaneously targeting two regions of the HCV 1b genome and the host cell receptor, CD81. The shRNAs directed against HCV IRES or NS5b regions were shown to be effective in inhibiting HCV replication in vitro (82 and 98%, respectively). No evidence of shRNA-related interferon production was observed. Vectors containing CD81 shRNA reduced cell surface expression up to 83% and reduced cell binding of HCV surface protein E2 up to 82% while not affecting levels of unrelated surface protein (Ber-EP4) or HCV replication. Double or triple shRNA vectors were independently effective in simultaneously reducing HCV replication, CD81 expression, and E2 binding. This study demonstrates lentiviral delivery of multiple shRNA, inhibiting HCV in a specific, IFN-independent, manner. The targeting of multiple viral and host cell elements simultaneously by RNAi could increase the potency of antiviral gene therapies.     

In ankylosing spondylitis serum interleukin-6 correlates with the degree of mobility restriction, but not with short-term changes in the variables for mobility

The aim of this study was to evaluate whether the serum concentration of interleukin-6 (IL-6) reflects disease activity in ankylosing spondylitis (AS). A group of 271 AS patients were enrolled in the study, 261 of whom completed the entire protocol (201 males, 60 females, median age of 53 years). Serum IL-6 was measured three times (I, baseline; II, after 10 – 12 days; III, after 17 – 24 days) during a 3- or 4-week treatment at the health resort. At the same times, the variables for mobility were measured, and the patients were asked to assess their complaints (score) in a self-styled questionnaire. The serum concentration of IL-6 correlated with the measurements of occiput-to-wall distance, cervical rotation, finger-floor distance and Schober sign, and with morning pain at all three evaluations. Comparisons between changes in IL-6 and changes in the variables (measures of mobility, scores of the questionnaires) did not reveal significant correlations. Present data would suggest that in AS the serum concentration of IL-6 indicates the degree of mobility restriction resulting from previous disease progression, but is not a reliable marker of current disease activity. Received: 10 February 1998 / Accepted: 9 July 1998     

Differential Mechanisms of Tenofovir and Tenofovir Disoproxil Fumarate Cellular Transport and Implications for Topical Preexposure Prophylaxis

Intravaginal rings releasing tenofovir (TFV) or its prodrug, tenofovir disoproxil fumarate (TDF), are being evaluated for HIV and herpes simplex virus (HSV) prevention. The current studies were designed to determine the mechanisms of drug accumulation in human vaginal and immune cells. The exposure of vaginal epithelial or T cells to equimolar concentrations of radiolabeled TDF resulted in over 10-fold higher intracellular drug levels than exposure to TFV. Permeability studies demonstrated that TDF, but not TFV, entered cells by passive diffusion. TDF uptake was energy independent but its accumulation followed nonlinear kinetics, and excess unlabeled TDF inhibited radiolabeled TDF uptake in competition studies. The carboxylesterase inhibitor bis-nitrophenyl phosphate reduced TDF uptake, suggesting saturability of intracellular carboxylesterases. In contrast, although TFV uptake was energy dependent, no competition between unlabeled and radiolabeled TFV was observed, and the previously identified transporters, organic anion transporters (OATs) 1 and 3, were not expressed in human vaginal or T cells. The intracellular accumulation of TFV was reduced by the addition of endocytosis inhibitors, and this resulted in the loss of TFV antiviral activity. Kinetics of drug transport and metabolism were monitored by quantifying the parent drugs and their metabolites by high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). Results were consistent with the identified mechanisms of transport, and the exposure of vaginal epithelial cells to equimolar concentrations of TDF compared to TFV resulted in ∼40-fold higher levels of the active metabolite, tenofovir diphosphate. Together, these findings indicate that substantially lower concentrations of TDF than TFV are needed to protect cells from HIV and HSV-2.     

Use of antibiotics during pregnancy

Pregnancy is occasionally complicated by infections that necessitate antibiotic therapy. When considering therapeutic options for pregnant women, both the physiologic changes of pregnancy and the prenatal effects of the drug must be weighed. Antibiotics should be selected with regard to the trimester of pregnancy. Some antibiotics are safe for use throughout pregnancy, while others are completely contraindicated. Choosing the proper antibiotic requires balancing the seriousness of the infection with the antibiotic's safety and antimicrobial activity.     

Case report of Lyell's syndrome

In Lyell's syndrome a toxic skin erythema is concerned which leads to the epidermal necrolysis and desquamation. An infantile and an adult form are differed; the latter is usually induced by medicaments. Two patients with apparently medicamentously induced Lyell's syndrome are presented. The evoking medicaments were penicillin and thioacetazone. The two patients showed a severe course with an extensive affection of the body surface. The therapy with glucocorticosteroids, electrolyte substitution and local treatment of the skin lesions was successful in the two cases.     

Persistent fatigue in liver transplant recipients: a two-year follow-up study

Background:  Fatigue after liver transplantation (LTx) is a major problem that is associated with lower daily functioning and health-related quality of life (HRQoL). This study aimed to assess changes over time in fatigue following LTx. We also examined daily functioning and HRQoL changes over time and assessed the influence of fatigue and changes in fatigue on daily functioning and HRQoL. We determined whether sleep quality, anxiety, and depression were associated with fatigue.
Methods:  We identified 70 LTx recipients who had previously participated in a cross-sectional study and reassessed them after two yr to determine changes in level of fatigue, daily functioning, and HRQoL. We also assessed sleep quality, anxiety, and depression after two yr.
Results:  Level of fatigue and level of daily functioning were unchanged at follow-up. HRQoL domains remained stable or worsened. Fatigue was a significant predictor of daily functioning and all HRQoL domains (p < 0.01). Change in fatigue was a significant predictor of daily functioning and the HRQoL domains of "physical functioning,""vitality," and "pain" (p < 0.05). Sleep quality, anxiety, and depression were associated with fatigue severity (r = 0.35 to r = 0.60, p < 0.05).
Conclusion:  This longitudinal study shows that fatigue is a chronic problem after LTx and that daily functioning and HRQoL do not improve over time. This study supports the need for intervention programs to address fatigue after LTx.     

Third generation anti-cyclic citrullinated peptide antibodies do not predict anti-TNF-α treatment response in rheumatoid arthritis

Objective of this study is to retrospectively compare the third generation anti-cyclic citrullinated peptide (anti-CCP3) test with the second generation (anti-CCP2) assay as markers of disease activity and predictors of clinical response in rheumatoid arthritis (RA) patients treated with TNF-α blocking agents. This study was performed in 42 RA patients treated either with infliximab (n = 11), etanercept (n = 7) or adalimumab (n = 24). Serum anti-CCP3 and anti-CCP2 levels were tested before and 6 months after starting a TNF-α blocking treatment using commercially available ELISA kits. Anti-CCP3 and anti-CCP2 antibody levels did not significantly change after 6 months of TNF-α blocking treatment. Furthermore, neither anti-CCP3 nor anti-CCP2 was useful to predict anti-TNF-α treatment response using receiving operating characteristic curve and logistic regression analyses. Both anti-CCP3 and anti-CCP2 are not differentially influenced by TNF-α blocking agents in RA patients and failed to predict anti-TNF-α treatment response.     

Nutritional impact on the plasma metabolome of rats

Metabolite profiling (metabolomics) elucidates changes in biochemical pathways under various conditions, e.g., different nutrition scenarios or compound administration. BASF and metanomics have obtained plasma metabolic profiles of approximately 500 compounds (agrochemicals, chemicals and pharmaceuticals) from 28-day rat studies. With these profiles the establishment of a database (MetaMap^®Tox) containing specific metabolic patterns associated with many toxicological modes of action was achieved. To evaluate confounding factors influencing metabolome patterns, the effect of fasting vs. non-fasting prior to blood sampling, the influence of high caloric diet and caloric restriction as well as the administration of corn oil and olive oil was studied for its influence on the metabolome. All mentioned treatments had distinct effects: triacylglycerol, phospholipids and their degradation product levels (fatty acids, glycerol, lysophosphatidylcholine) were often altered depending on the nutritional status. Also some amino acid and related compounds were changed. Some metabolites derived from food (e.g. alpha-tocopherol, ascorbic acid, beta-sitosterol, campesterol) were biomarkers related to food consumption, whereas others indicated a changed energy metabolism (e.g. hydroxybutyrate, pyruvate). Strikingly, there was a profound difference in the metabolite responses to diet restriction in male and female rats. Consequently, when evaluating the metabolic profile of a compound, the effect of nutritional status should be taken into account.     

Reproducibility and robustness of metabolome analysis in rat plasma of 28-day repeated dose toxicity studies

BASF has developed a rat plasma metabolomics database (MetaMap^®Tox) containing the metabolome of more than 500 chemicals, agrochemicals and drugs, for which the toxicity is well known, derived from 28-day repeated dose toxicity studies in rats. The quality/reproducibility of data was assessed by comparing the metabolome of 16 reference compounds tested at least twice under identical experimental conditions at three time points (day 7, day 14 and day 28). Statistical correlation analysis showed that the repeated treatment induced very similar changes to the metabolome. For all repetitions the modes of action of the compounds were always correctly identified. Moreover, when compared against the metabolome of all compounds available in the MetaMap^®Tox database, the repetitions showed in most cases the highest degree of overall similarity with the metabolome of the original study. In addition, we also evaluated the robustness of our metabolomics technique, displayed by constancy of variability in control groups over time. Based on these results, it can be concluded, that metabolomics can reproducibly be applied during toxicological in vivo testing in rats under the conditions applied here.