Effect of Topical Anaesthetics on Interstitial Colloid Osmotic Pressure in Human Subcutaneous Tissue Sampled by Wick Technique

To measure colloid osmotic pressure in interstitial fluid (COP(i)) from human subcutaneous tissue with the modified wick technique in order to determine influence of topical application of anaesthetics, dry vs. wet wick and implantation time on COP(i).In 50 healthy volunteers interstitial fluid (IF) was collected by subcutaneous implantation of multi-filamentous nylon wicks. Study subjects were allocated to two groups; one for comparing COP(i) obtained from dry and saline soaked wicks, and one for comparing COP(i) from unanaesthetized skin, and skin after application of a eutectic mixture of local anaesthetic (EMLA®, Astra Zeneca) cream. IF was sampled from the skin of the shoulders, and implantation time was 30, 60, 75, 90 and 120 min. Colloid osmotic pressure was measured with a colloid osmometer. Pain assessment during the procedure was compared for EMLA cream and no topical anaesthesia using a visual analogue scale (VAS) in a subgroup of 10 subjects.There were no significant differences between COP(i) obtained from dry compared to wet wicks, except that the values after 75 and 90 min. were somewhat higher for the dry wicks. Topical anaesthesia with EMLA cream did not affect COP(i) values. COP(i) decreased from 30 to 75 min. of implantation (23.2 ± 4.4 mmHg to 19.6 ± 2.9 mmHg, p = 0.008) and subsequently tended to increase until 120 min. EMLA cream resulted in significant lower VAS score for the procedure.COP(i) from subcutaneous tissue was easily obtained and fluid harvesting was well tolerated when topical anaesthetic was used. The difference in COP(i) assessed by dry and wet wicks between 75 min. and 90 min. of implantation was in accordance with previous reports. The use of topical analgesia did not influence COP(i) and topical analgesia may make the wick technique more acceptable for subjects who dislike technical procedures, including children.ClinicalTrials.gov NCT01044979

Istraživanja 3,4-diaril-1,2,5-oksadiazola i njihovih N-oksida: Potraga za boljim COX-2 inhibitorima

A series of 3,4-diaryl-1,2,5-oxadiazoles and 3,4-diaryl-1,2,5-oxadiazole N-oxides were prepared and evaluated for COX-2 and COX-1 binding affinity in vitro and for anti-inflammatory activity by the rat paw edema method. p-Methoxy (p-OMe) substituted compounds 9, 21, 34, 41, 42 showed COX-2 enzyme inhibition higher than that showed by compounds with other substituents. 3,4-Di(4-methoxyphenyl)-1,2,5-oxadiazole N-oxide (42) showed COX-2 enzyme inhibition of 54% at 22 µmol L-1 and COX-1 enzyme inhibition of 44% at 88 µmol L-1 concentrations, but showed very low in vivo anti-inflammatory activity. Its deoxygenated derivative (21) showed lower COX-2 enzyme inhibition (26% at 22 µmol L-1) and higher COX-1 enzyme inhibition (53% at 88 µmol L-1) but marked in vivo anti-inflammatory activity (71% at 25 mg kg-1) vs. celecoxib (48% at 12.5 mg kg-1). Molecular modeling (docking) studies showed that the methoxy group is positioned in the vicinity of the COX-2 secondary pocket and it also participates in hydrogen bonding interactions in the COX-2 active site. These preliminary studies suggest that the p-methoxy (p-OMe) group in one of benzene rings may give potentially active leads in this series of oxadiazole/N-oxides.Sintetizirana je serija 3,4-diaril-1,2,5-oksadiazola i 3,4-diaril-1,2,5-oksadiazol N-oksida i ocijenjena njihova sposobnost vezivanja na COX-2 i COX-1 in vitro i protuupalno djelovanje na edem šape štakora. Spojevi sa p-metoksi (p-OMe) supstituentom 9, 21, 34, 41, 42 bolje su inhibirali COX-2 nego ostali spojevi. 3,4-Di(4-metoksifenil)-1,2,5-oksadiazol N-oksid (42) inhibirao je COX-2 za 54% u koncentraciji od 22 µmol L-1, a COX-1 za 44% u koncentraciji 88 µmol L-1, ali je in vivo slabo djelovao protuupalno. Njegov deoksigenirani derivat 21 pokazao je slabiju inhibiciju COX-2 enzima (26% u koncentraciji 22 µmol L-1) i jaču inhibiciju COX-1 (71% u koncentraciji 25 mg kg-1) što je bolje od standarda celekoksiba (48% u koncentraciji 12,5 mg kg-1). Molekularno je modeliranje pokazalo da je metoksi skupina smještena u blizini sekundarnog džepa na enzimu COX-2 i da utječe na vodikove veze interakcija na aktivnom mjestu COX-2. Ova preliminarna istraživanja sugeriraju da bi se u seriji oksadiazol/N-oksida mogao naći predvodni spoj s p-metoksi skupinom na benzenskom prstenu

Human Macrophages Infected with a High Burden of ESAT-6-Expressing M. tuberculosis Undergo Caspase-1- and Cathepsin B-Independent Necrosis

Mycobacterium tuberculosis (Mtb) infects lung macrophages, which instead of killing the pathogen can be manipulated by the bacilli, creating an environment suitable for intracellular replication and spread to adjacent cells. The role of host cell death during Mtb infection is debated because the bacilli have been shown to be both anti-apoptotic, keeping the host cell alive to avoid the antimicrobial effects of apoptosis, and pro-necrotic, killing the host macrophage to allow infection of neighboring cells. Since mycobacteria activate the NLRP3 inflammasome in macrophages, we investigated whether Mtb could induce one of the recently described inflammasome-linked cell death modes pyroptosis and pyronecrosis. These are mediated through caspase-1 and cathepsin-B, respectively. Human monocyte-derived macrophages were infected with virulent (H37Rv) Mtb at a multiplicity of infection (MOI) of 1 or 10. The higher MOI resulted in strongly enhanced release of IL-1β, while a low MOI gave no IL-1β response. The infected macrophages were collected and cell viability in terms of the integrity of DNA, mitochondria and the plasma membrane was determined. We found that infection with H37Rv at MOI 10, but not MOI 1, over two days led to extensive DNA fragmentation, loss of mitochondrial membrane potential, loss of plasma membrane integrity, and HMGB1 release. Although we observed plasma membrane permeabilization and IL-1β release from infected cells, the cell death induced by Mtb was not dependent on caspase-1 or cathepsin B. It was, however, dependent on mycobacterial expression of ESAT-6. We conclude that as virulent Mtb reaches a threshold number of bacilli inside the human macrophage, ESAT-6-dependent necrosis occurs, activating caspase-1 in the process

The oxysterol 27-hydroxycholesterol increases β-amyloid and oxidative stress in retinal pigment epithelial cells

<p>Abstract</p> <p>Background</p> <p>Alzheimer's disease (AD) and age-related macular degeneration (AMD) share several pathological features including β-amyloid (Aβ) peptide accumulation, oxidative damage, and cell death. The causes of AD and AMD are not known but several studies suggest disturbances in cholesterol metabolism as a culprit of these diseases. We have recently shown that the cholesterol oxidation metabolite 27-hydroxycholesterol (27-OHC) causes AD-like pathology in human neuroblastoma SH-SY5Y cells and in organotypic hippocampal slices. However, the extent to which and the mechanisms by which 27-OHC may also cause pathological hallmarks related to AMD are ill-defined. In this study, the effects of 27-OHC on AMD-related pathology were determined in ARPE-19 cells. These cells have structural and functional properties relevant to retinal pigmented epithelial cells, a target in the course of AMD.</p> <p>Methods</p> <p>ARPE-19 cells were treated with 0, 10 or 25 μM 27-OHC for 24 hours. Levels of Aβ peptide, mitochondrial and endoplasmic reticulum (ER) stress markers, Ca²⁺homeostasis, glutathione depletion, reactive oxygen species (ROS) generation, inflammation and cell death were assessed using ELISA, Western blot, immunocytochemistry, and specific assays.</p> <p>Results</p> <p>27-OHC dose-dependently increased Aβ peptide production, increased levels of ER stress specific markers caspase 12 and gadd153 (also called CHOP), reduced mitochondrial membrane potential, triggered Ca²⁺dyshomeostasis, increased levels of the nuclear factor κB (NFκB) and heme-oxygenase 1 (HO-1), two proteins activated by oxidative stress. Additionally, 27-OHC caused glutathione depletion, ROS generation, inflammation and apoptotic-mediated cell death.</p> <p>Conclusions</p> <p>The cholesterol metabolite 27-OHC is toxic to RPE cells. The deleterious effects of this oxysterol ranged from Aβ accumulation to oxidative cell damage. Our results suggest that high levels of 27-OHC may represent a common pathogenic factor for both AMD and AD.</p

Chemical and protein structural basis for biological crosstalk between PPARα and COX enzymes

We have previously validated a probabilistic framework that combined computational approaches for predicting the biological activities of small molecule drugs. Molecule comparison methods included molecular structural similarity metrics and similarity computed from lexical analysis of text in drug package inserts. Here we present an analysis of novel drug/target predictions, focusing on those that were not obvious based on known pharmacological crosstalk. Considering those cases where the predicted target was an enzyme with known 3D structure allowed incorporation of information from molecular docking and protein binding pocket similarity in addition to ligand-based comparisons. Taken together, the combination of orthogonal information sources led to investigation of a surprising predicted relationship between a transcription factor and an enzyme, specifically, PPARα and the cyclooxygenase enzymes. These predictions were confirmed by direct biochemical experiments which validate the approach and show for the first time that PPARα agonists are cyclooxygenase inhibitors