Workplace drug testing on urine samples: evidence for improving efficacy of a first-level screening programme.

BACKGROUND: Previous reports revealed poor performance in identifying drugs of abuse users through first-level workplace drug testing (WDT), based on urine samples. In a cross-sectional study, we evaluated: (i) the effect of creatinine normalization of drug values from diluted urine samples (creatinine levels ≤ 20 mg/dL) on the prevalence of drug users; (ii) the independent procedure-related predictors of positivity and dilution. METHODS: Workers' urine samples were collected at the workplace or at our certified laboratory between 2008 and 2012. All samples were analysed for drugs of abuse by immuno-enzymatic method in our laboratory, according to the Italian WDT law. Detectable drugs of abuse concentrations lower than the positive cutoff values were normalized based on mean levels of urinary creatinine. Detectable concentrations of drugs were confirmed by GC/MS. Multivariate logistic regression was used to detect independent procedure-related predictors of positive and diluted urine samples. RESULTS: Of the 3080 urine samples screened, 51 (1.7%) were found positive for some drugs of abuse (26 cannabinoids and 16 cocaine) and 116 (3.8%) were diluted. Seventeen out of 23 diluted urine samples with detectable concentrations of cannabinoids or cocaine were found positive after urine creatinine normalization and GC/MS confirmed both negative and positive results. This increased the percentage of positivity for cannabinoids and cocaine from 1.35% to 2.09% (+55%, p=0.0005), which is closer to the expected prevalence of drug users based on Italian self-reported surveys. Collection of samples in the laboratory was an independent predictor of positivity (OR=2.33, 95%CI 1.27-4.28) and diluted urine sample (OR=1.65, 95%CI 1.04-2.61). CONCLUSIONS: Efficacy of first-level WDT could be improved by well-controlled pre-analytical procedures and urine creatinine normalization of detected concentrations of drugs of abuse.

Effect of the lipopolysaccharide antagonist Planktothrix sp. FP1 cyanobacterial extract on human polymorphonuclear leukocytes.

CyP is a lipopolysaccharide (LPS)-like molecule extracted from the freshwater cyanobacterium Oscillatoria planktothrix FP1, which has been reported to be a potent competitive inhibitor of bacterial LPS. In the present study the ability of CyP to affect human polymorphonuclear leukocyte (PMN) function was investigated. PMNs were isolated from venous blood by standard density-gradient centrifugation. Cell migration was measured by use of the Boyden chamber assay. Interleukin (IL)-8 and tumor necrosis factor (TNF)-α production was measured using a sandwich-type enzyme-linked immunosorbent assay. PMN intracellular reactive oxygen species (ROS) levels were assessed by the use of a fluorescent probe coupled to spectrophotometry. CyP 10-100 µg/ml was chemotactic for PMNs without affecting the chemotactic response to either E. coli LPS or N-formyl-Met-Leu-Phe (fMLP). CyP per se did not affect PMN production of either IL-8 or TNF-α, but concentration-dependently reduced LPS-induced production of both cytokines. On the contrary, CyP had no effect either on fMLP-induced production of IL-8 or on PMN oxidative burst (at rest and after stimulation with fMLP), a response which is known to be independent from LPS-operated pathways. In human PMNs CyP behaves as a selective and effective LPS antagonist. These findings support the therapeutic potential of CyP in endotoxin-dependent disease.

Scanning electron microscopy examination of endothelium morphology in human carotid plaques.

In recent years, experimental and clinical evidence has been provided regarding endothelial dysfunction and its contribution to the inflammatory process leading to atherosclerotic plaque formation. All the techniques employed direct to the study of endothelial dysfunction, however usually require isolation of endothelial cells and therefore tissue manipulation and destruction, with subsequent loss of information regarding morphology and topographical distribution of the lesions. By means of scanning electron microscopy (SEM), we have investigated the characteristics of the endothelial layer in carotid specimens obtained from subjects undergoing carotid endarterectomy. Carotid specimens obtained from 6 patients undergoing carotid endarterectomy for stenosis> or =70% were fixed, prepared and examined by use of SEM in a direct mode. All the plaque specimens showed increased thickness of subendothelial connective tissue with respect to post-mortem tissue obtained from an healthy subject. Endothelial layers were typically detached from the basal lamina surface and infiltrating cells (mainly erythrocytes and, possibly, monocytes) could be identified. Endothelial cells in proximity of the plaques had irregular shape, with prominent nuclei. In several areas, the endothelial layer was completely absent and basal lamina completely uncovered. In the present study, by using SEM analysis, the morphological features of dysfunctional endothelium in human carotid plaques were extensively documented at the ultrastructural level. SEM is a powerful investigational technique which allows tridimensional examination of specimens without disruption of the originary morphology.

Immunomodulatory activity of the lignan 7-hydroxymatairesinol potassium acetate (HMR/lignan) extracted from the heartwood of Norway spruce (Picea abies).

The pharmacological profile of the lignan 7-hydroxymatairesinol (HMR/lignan, HMR) includes chemopreventive effects, antioxidant properties, and mild proestrogenic activity. The present study was devised to investigate the effects of HMR on THP-1 cells, an established model of human monocytes, and on human polymorphonuclear leukocytes (PMNs). In THP-1 cells, HMR concentration-dependently reduced LPS-stimulated tumor necrosis factor (TNF)-alpha secretion in the supernatant. HMR at low, sub-muM concentrations also reduced TNF-alpha mRNA, which was however enhanced by supra-muM concentrations of HMR. In human PMNs, HMR concentration-dependently reduced ROS production induced by either N-formyl-Met-Leu-Phe, phorbol myristate acetate or angiotensin II, as well as interleukin-8 production induced by either N-formyl-Met-Leu-Phe or angiotensin II. Results indicate that HMR is an effective inhibitor of both monocytic THP-1 cells and of human PMNs and warrant further studies to assess their relevance for the prevention and treatment of several conditions characterized by chronic systemic inflammation.

Simvastatin treatment in subjects at high cardiovascular risk modulates AT1R expression on circulating monocytes and T lymphocytes.

OBJECTIVE: Angiotensin II, through the activation of angiotensin II type 1 receptors, plays a crucial role in atherosclerosis. Statins may interfere with the effects of angiotensin II. METHODS: We have investigated the expression of angiotensin II type 1 receptor, angiotensin II type 2 receptor and angiotensinogen on circulating monocytes and T-lymphocytes from subjects at high risk for vascular events before and during simvastatin treatment, and healthy controls. In-vitro experiments were also performed to assess the ability of simvastatin to interfere with angiotensin II signalling. RESULTS: In comparison with controls, high-risk subjects had similar angiotensin II type 1 receptor expression on the cell membranes but significantly higher angiotensin II type 1 receptor mRNA levels at least in monocyte subsets whereas their expression on T cells was similar. Angiotensin II type 2 receptor mRNA expression was higher than controls in both monocytes and T lymphocytes. No differences were observed in angiotensinogen expression on monocytes while T lymphocytes of high-risk subjects show higher expression. One-month treatment of high-risk subjects with simvastatin resulted in a reduction of angiotensin II type 1 receptor mRNA without affecting angiotensin II type 2 receptor whereas angiotensinogen mRNA expression was reduced at least in monocytes. Incubation in vitro with simvastatin reduces the expression of angiotensin II type 1 receptor mRNA levels on monocytes from untreated subjects. CONCLUSION: Simvastatin induces down-regulation of the angiotensin II type 1 receptor, interferes with angiotensin II activity in immune cells and contributes to the anti-inflammatory profile of statins that can explain the therapeutic effects of these drugs.

Angiotensin II type 1 receptor expression in polymorphonuclear leukocytes from high-risk subjects: changes after treatment with simvastatin.

Statins may directly interfere with the effects of angiotensin (Ang) II, which is a key player in the pathogenesis of atherosclerosis (ATH). Ang II promotes a wide array of detrimental processes including a prominent proinflammatory effect, increasingly regarded as a target for therapeutic intervention. Because the proinflammatory effects of Ang II are exerted mainly through the activation of Ang II type 1 receptors (AT1Rs) the present study was devised to investigate by means of real-time polymerase chain reaction (PCR) and flow cytometry techniques the expression of such receptors on circulating polymorphonuclear leukocytes (PMNs) from subjects at high risk for vascular events before and during treatment with simvastatin and in sex- and age-matched healthy controls. In vitro experiments were also performed to assess the ability of simvastatin to interfere with Ang II signaling in human PMNs. In comparison to controls, high-risk subjects had similar AT1R expression on the cell membranes but significantly higher AT1R messenger ribonucleic acid (mRNA) levels. Treatment of high-risk subjects with simvastatin for 30 days resulted in a reduction of AT1R mRNA down to the levels of cells from healthy subjects. In vitro, Ang II-induced activation of the guanosine triphosphate (GTP)-binding protein Rac 1 in human PMNs was inhibited by simvastatin. In conclusion, simvastatin induces downregulation of AT1R expression, interferes with Ang II activity in PMNs, and contributes to the antiinflammatory profile of statins that can explain the therapeutic effects of these drugs.