Fas ligand downregulation with antisense oligonucleotides in cells and in cultured tissues of normal skin epidermis and basal cell carcinoma

Fas ligand (FasL), a member of the tumor necrosis factor family, induces apoptosis upon interaction with Fas-receptor-expressing cells. FasL normally plays an important immune regulatory role, but it can also cause severe skin diseases if overexpressed and it may serve some tumors for immune evasion. Thus, in situ inhibition of FasL expression with antisense oligonucleotides in patients may be a novel approach to overcome its pathogenic role. We designed and evaluated 15 phosphorothioate antisense oligonucleotides directed against different regions of the human FasL mRNA. They exhibited different inhibitory activities on FasL expression in HEK293 cells. The most potent antisense oligonucleotide, ASO8, specifically downregulated 90% FasL expression at the protein level and 80% at the mRNA level. FasL downregulation reduced the effector function of HEK293 cells toward Fas receptor positive target cells. Further studies demonstrated that ASO8 efficiently inhibited FasL synthesis in split skin and basal cell carcinoma tissue. Our results show that the modulation of FasL expression by antisense oligonucleotides is possible in cells as well as tissue and indicate that antisense oligonucleotides may provide a promising strategy for the therapy of FasL-mediated disorders.     

Level of agreement between physical activity levels measured by ActiHeart and the International Physical Activity Questionnaire in persons with intellectual disability

Purpose: The purpose of this study was to determine the level of agreement between objective physical activity (PA) (ActiHeart^®) and subjective proxy-respondent International Physical Activity Questionnaire-short version (IPAQ-S) data in adults with intellectual disabilities (IDs).

Method: Fifty-eight participants wore ActiHeart^® monitors for seven consecutive days. Caregivers of each participant completed the IPAQ-S on behalf of the participant. Total PA, time spent in light, moderate, and vigorous activity as well as time spent being sedentary were assessed by the IPAQ-S and the ActiHeart^®. Results were compared by means of correlation analyses. The level of agreement was presented with Bland–Altman plots.

Results: Objective PA (ActiHeart^®) was higher (225.57?±?91.96?min/week) than IPAQ-S PA reported by care-givers (177.06?±?309.17?min/week). Weak significant correlations were observed between the ActiHeart^® and IPAQ-S instruments for sedentary behavior (r?=?0.31; p?=?0.04); no significant correlations for light (r=??0.04; p?=?0.8), moderate (r=??0.07; p?=?0.63), or vigorous PA (r=??0.2; p?=?0.18) were found. Limited agreement between objectively determine PA (ActiHeart^®) and IPAQ-S was found.

Conclusion: IPAQ-S is inaccurate when determining PA in persons with ID as it significantly underestimates the true levels of PA in this cohort.

• Implications for Rehabilitation

• Persons with intellectual disability (ID) report insufficient physical activity for health benefits.

• Physical activity is often determined by means of subjective proxy reporting.

• Objective physical activity measurements by means of combined heart rate and accelerometer are necessary to determine accurate levels of physical activity in persons with ID.

• Exercise interventions should be based on objective physical activity measurements.

     

Role of Peptide YY in Intestinal and Gallbladder Motility in Dogs

The purpose of this study was to characterize the action of exogenous PYY, an ileocolonic peptide released by fatty meal, and that released by Heal perfusion with oleate on intestinal and gallbladder motility patterns and the posssible role of the adrenergic pathway in this action. Dogs were equipped with chronic duodenal electrodes for recording myoelectric activity and with a cannula in the gallbladder fundus for measuring the gallbladders intraluminal pressure and volume and calculating its motility index (MI) and emptying rate. After intravenous infusion of PYY, there was a dose-dependent prolongation of the migrating motor complex (MMC) interval and almost complete abolition of the contractions and emptying of gallbladder during the duodenal activity front. After meat feeding or during intravenous infusion of cerulein, 50 pmol/(kg · h), the MMC was interrupted and replaced by irregular spike activity, accompanied by a marked increase in the gallbladder MI and about 80% to 90% reduction of its volume. PYY, 200 pmol/(kg · h), reduced significantly the meal- or cerulein-induced duodenal spike activity but failed to affect the MI and volume of the gallbladder. Similar changes in fasted and fed patterns of motility were observed after Heal oleate (16 mM/h), producing plasma PYY levels in a range similar to that observed after infusion of exogenous PYY. The inhibitory effects of PYY or Heal fat on intestinal myoelectric activity were reversed in part by α-adrenergic blockade (phentolamine). We conclude that exogenous PYY or endogenous hormone released by Heal oleate inhibits the interdigestive and postprandial motility pattern of the small bowel but does not affect gallbladder motility, and that the inhibition of intestinal motility involves, at least in part, the adrenergic pathway.     

Interactions between two enantiomers of losigamone and conventional antiepileptic drugs in the mouse maximal electroshock model--an isobolographic analysis

The aim of this study was the isobolographic evaluation of interactions between two enantiomers of losigamone, AO-242 [(+)-5(R)-alpha(S)-5-(2-chlorophenylhydroxymethyl)-4-methoxy-2(5H)-furanone] and AO-294 [(-)-5(S)-alpha(R)-5-(2-chlorophenylhydroxymethyl)-4-methoxy-2(5H)-furanone], and valproate, carbamazepine, phenytoin, or phenobarbital in the maximal electroshock test in mice. Both enantiomers interacted additively with conventional antiepileptic drugs at all studied fixed dose ratios (1:3, 1:1, 3:1). Furthermore, AO-242, AO-294 and antiepileptics applied alone, as well as combinations of enantiomers and antiepileptics did not affect motor performance in the chimney test. Significant impairment of long-term memory (passive-avoidance task) was noted only in the case of valproate alone, given at the dose equal to its median effective dose (ED(50)) against maximal electroshock. All other antiepileptics and their combinations with AO-242 or AO-294 did not impair memory of mice. Enantiomers did not affect the brain concentrations of antiepileptic drugs, indicating a pharmacodynamic nature of the observed interactions. In conclusion, the present results suggest both AO-242 and AO-294 as promising candidate drugs in the add-on therapy of refractory epilepsy.     

Deep venous thrombosis and pulmonary embolism in trauma patients: an overstatement of the problem?

Deep venous thrombosis (DVT) and pulmonary embolism (PE) affect high-risk trauma patients (HRTP). Accurate incidence and clinical importance of DVT and PE in HRPT may be overstated. We performed a ten-year retrospective analysis of HRTP of the Pennsylvania Trauma Outcome Study. High-risk factors (HRF) included pelvic fracture (PFx), lower extremity fracture (LEFx), severe head injury (CHI) (AIS - head > or =3), and spinal cord injury. HRF alone or in combination, age, Injury Severity Score (ISS), and Glasgow Coma Score (GCS) were examined for association with DVT/PE. A total of 73,419 HRTP were included: 1377 (1.9%) had DVT, 365 (0.5%) had PE. The incidence of DVT in level I trauma centers was 2.2 per cent and was 1.5 per cent in level II centers. The lowest incidence of DVT was 1.3 per cent for isolated LEFx; highest was 5.4% for combined PFx, LEFx, and CHI. Variables associated with DVT included age, ISS, and GCS (all P < 0.001). In logistic regression analysis, only ISS was consistently predictive for DVT and PE. Though increased during the past decade, the overall incidence of DVT in HRTP remains below 3 per cent. Only the combination of multiple injuries or an ISS >30 result in DVT incidence of > or =5 per cent. We believe that current guidelines for screening for DVT may need to be reevaluated.     

The Human Tongue Slows Down to Speak: Muscle Fibers of the Human Tongue

Little is known about the specializations of human tongue muscles. In this study, myofibrillar adenosine triphosphatase (mATPase) histochemical staining was used to study the percentage and distribution of slow twitch muscle fibers (slow MFs) within tongue muscles of four neurologically normal human adults and specimens from a 2‐year‐old human, a newborn human, an adult with idiopathic Parkinson's disease (IPD), and a macaque monkey. The average percentage of slow MFs in adult and the 2‐year‐old muscle specimens was 54%, the IPD was 45%, while the neonatal human (32%) and macaque monkey (28%) had markedly fewer slow MFs. In contrast, the tongue muscles of the rat and cat have been reported to have no slow MFs. There was a marked spatial gradient in the distribution of slow MFs with the highest percentages found medially and posteriorly. Normal adult tongue muscles were found to have a variety of uniquely specialized features including MF‐type grouping (usually found in neuromuscular disorders), large amounts of loose connective tissue, and short branching MFs. In summary, normal adult human tongue muscles have by far the highest proportion of slow MFs of any mammalian tongue studied to date. Moreover, adult human tongue muscles have multiple unique anatomic features. As the tongue shape changes that are seen during speech articulation are unique to humans, we hypothesize that the large proportion of slow MFs and the anatomical specializations observed in the adult human tongue have evolved to perform these movements. Anat Rec, 296:1615–1627, 2013. © 2013 Wiley Periodicals, Inc.     

Kinetics of the anionic polymerization of ϵ-caprolactone with K+ · (dibenzo-18-crown-6 ether) counterion. Propagation via macroions and macroion pairs

Following our earlier work on the polymerization of lactones involving crowned cations, kinetics of the anionic polymerization of ?-caprolactone (?CL) with K⁺ · (dibenzo-18-crown-6 ether) (K⁺DB18C6) counterion was studied calorimetrically in THF solution in the temperature range from 0 to 20°C. Dissociation constants of CH₃(CH₂)₅O^?K⁺DB18C6, modelling the active centers, were determined conductometrically: K_D (20°C) = 7,7 · 10^?5 mol · dm^?3, ΔH = 9,3 ± 0,2 kJ · mol^?1, ΔS = ?47 ± 2J · mol^?1 · K^?1. From kinetic measurements and from measurements of the dissociation constant of CH₃(CH₂)₅O^? K⁺DB18C6, rate constants of propagation via macroions and via macroion pairs were determined. Activation parameters for propagation via these species are equal to: ΔH = 39,2 ± 0,2 kJ · mol^?1, ΔS = ?63 ± 1 J · mol^?1 · K^?1, ΔH = 13,7 ± 0,1 kJ · mol^?1, ΔS = ?185 ± 2 J · mol^?1 · K^?1. At 20°C, k = 3,50 · 10² dm³ · mol^?1 · s^?1 and k = 5,2 dm³ · mol^?1 · s^?1. Due to the large difference of ΔH^≠ for propagation via macroions and macroion pairs (vide supra), the isokinetic point (k = k) would appear at ?65°C.     

Functional and morphological aspects of Helicobacter pylori-induced gastric cancer in Mongolian gerbils

BACKGROUND: Helicobacter pylori infection of Mongolian gerbils is an established model of gastric carcinogenesis, but gastric secretory aspects of this carcinogenesis have not been studied. METHODS: The effects of single intragastric inoculation of gerbils with H. pylori strain (cagA+ vacA+, 5 x 10(6) CFU/ml) or vehicle (saline) were examined at 1, 2, 4, 6, 9, 12 and 30 weeks from inoculation. Gastric morphology, the presence of H. pylori using the rapid urease test, the density of H. pylori and 16S rRNA and the plasma gastrin and somatostatin were determined. RESULTS: H. pylori was detected in gastric mucosa in all infected animals. Basal gastric acid in gerbils was reduced by about 50% after H. pylori inoculation. Early lesions seen at 4 weeks after H. pylori inoculation consisted of chronic gastritis with thickened mucosal folds, oedema, congestion and mucosal lymphocytic infiltration. Adenomatous hyperplasia with cellular atypia with increased mitotic activity and the formation of apoptotic bodies and visible erosions and ulcerations were observed at 12-30 weeks after inoculation. The atypical gastric glands were situated 'back-to-back', suggesting gastric pre-cancer. The gastric blood flow in H. pylori-infected gerbils was significantly lower than that in the controls. Six- to seven-fold increase in plasma gastrin levels combined with significant fall in gastric somatostatin contents and the intraepithelial neoplasia were noticed in gerbils at all tested periods. CONCLUSION: H. pylori-infection in gerbils resulted in gastric pre-cancer associated with functional changes, such as suppression of gastric secretion and impairment of both gastric mucosal microcirculation and the gastrin-somatostatin link.