Chiara Chiabrando

Estimating community drug abuse by wastewater analysis.

Background The social and medical problems of drug abuse are a matter of increasing global concern. To tackle drug abuse in changing scenarios, international drug agencies need fresh methods to monitor trends and patterns of illicit drug consumption. Objective We tested a sewage epidemiology approach, using levels of excreted drug residues in wastewater, to monitor collective use of the major drugs of abuse in near real time. Methods Selected drug target residues derived from use of cocaine, opiates, cannabis, and amphetamines were measured by mass spectrometry in wastewater collected at major sewage treatment plants in Milan (Italy), Lugano (Switzerland), and London (United Kingdom). The amounts of drug residues conveyed to the treatment plants, reflecting the amounts collectively excreted with urine, were used to estimate consumption of the active parent drugs. Results Reproducible and characteristic profiles of illicit drug use were obtained in the three cities, thus for the first time quickly revealing changes in local consumption (e.g., cocaine consumption rose significantly on weekends in Milan). Profiles of local drug consumption based on waste-water measurements are in line with national annual prevalence estimates. Conclusions Patterns and trends of drug abuse in local communities can be promptly monitored by this tool, a convenient new complement to more complex, lengthy survey methods. In principle, searching the sewage for excreted compounds relevant to public health issues appears to have the potential to become a convenient source of real-time epidemiologic information.

Tumor-Conditioned Macrophages Secrete Migration-Stimulating Factor: A New Marker for M2-Polarization, Influencing Tumor Cell Motility

Tumor-associated macrophages (TAMs) are key orchestrators of the tumor microenvironment directly affecting neoplastic cell growth, neoangiogenesis, and extracellular matrix remodeling. In turn, the tumor milieu strongly influences maturation of TAMs and shapes several of their features. To address the early macrophage (Mϕ) differentiation phase in a malignant context, we mimicked a tumor microenvironment by in vitro coculturing human blood monocytes with conditioned media from different cancer cell lines. Only 2 out of 16 tumor cell lines induced Mϕ differentiation due to secreted M-CSF isoforms, including high molecular mass species. A global gene profiling of tumor-conditioned Mϕ was performed. Comparison with other datasets (polarized M1-Mϕ, M2-Mϕ, and TAMs isolated from human tumors) highlighted the upregulation of several genes also shared by TAM and M2-polarized Mϕ. The most expressed genes were selenoprotein 1, osteoactivin, osteopontin, and, interestingly, migration-stimulating factor (MSF), a poorly studied oncofoetal isoform of fibronectin. MSF (present in fetal/cancer epithelial and stromal cells but not in healthy tissues) was never identified in Mϕ. MSF production was confirmed by immunohistochemistry in human TAMs. MSF was induced by M-CSF, IL-4, and TGFβ but not by proinflammatory stimuli. RNA and protein analysis clearly demonstrated that it is specifically associated with the M2 polarization of Mϕ. Tumor-conditioned Mϕ-derived MSFs strongly stimulated tumor cell migration, thus contributing to the motile phenotype of neoplastic cells. In conclusion, MSF is a new molecule associated with the M2 polarization of Mϕ and expressed by TAMs. Its biological function may contribute to Mϕ-mediated promotion of cancer cell invasion and metastasis.

Measurement of urinary 8-epi-prostaglandin f2α, a novel index of lipid peroxidation in vivo, by immunoaffinity extraction/gas chromatography-mass spectrometry. Basal levels in smokers and nonsmokers

8-Epi-prostaglandin F2alpha (8-epi-PGF2alpha) is an F2-isoprostane recently identified as a marker of free radical-catalyzed lipid peroxidation in vivo and potential mediator of oxidative damage. Currently, endogenous 8-epi-PGF2alpha is measured by gas chromatography-mass spectrometry after lengthy sample preparation. We extracted and purified 8-epi-PGF2alpha in one step from biological samples on immunoaffinity columns prepared with an anti-8-epi-PGF2alpha antiserum, raised in our laboratory. Quantitation was done by stable-isotope dilution gas chromatography/negative-ion chemical ionization mass spectrometry, with selected ion recording. Carboxylate anions of the pentafluorobenzyl ester trimethylsilyl ether derivative of 8-epi-PGF2alpha and [2H4]8-epi-PGF2alpha were monitored (m/z 569 and 573). Basal urinary excretion of 8-epi-PGF2alpha can be accurately and rapidly measured by this method. Under normal conditions rats (n = 30) excreted 2.18 +/- 0.68 ng/24 h. In healthy nonsmoking young volunteers, urinary excretion of 8-epi-PGF2alpha, measured three times on alternate days, was fairly constant (CV 2-10%). Nonsmokers excreted significantly less 8-epi-PGF2alpha than age-matched smokers (8.08 +/- 2.3 vs. 18.40 +/- 4.77 ng/h/1.73 m2; n = 6; p < 0.005), as reported by others using different methods.

Mass spectrometric analysis of illicit drugs in wastewater and surface water.

Residues of illicit drugs have been recently found in urban wastewater and surface water. Their levels reflect the amount of drugs collectively excreted by consumers and can therefore be used to estimate drug abuse. An overview of the most widely used illicit drugs and of the analytical methods used for their detection in wastewater and surface water is presented here. Solid-phase extraction and high performance liquid chromatography-tandem mass spectrometry are the techniques that have been used for these investigations. Instrumental conditions and fragmentation patterns of illicit drugs and their metabolites are described.

Proteasomal Processing of Albumin by Renal Dendritic Cells Generates Antigenic Peptides

The role of dendritic cells (DC) that accumulate in the renal parenchyma of non-immune-mediated proteinuric nephropathies is not well understood. Under certain circumstances, DC capture immunologically ignored antigens, including self-antigens, and present them within MHC class I, initiating an autoimmune response. We studied whether DC could generate antigenic peptides from the self-protein albumin. Exposure of rat proximal tubular cells to autologous albumin resulted in its proteolytic cleavage to form an N-terminal 24-amino acid peptide (ALB1-24). This peptide was further processed by the DC proteasome into antigenic peptides that had binding motifs for MHC class I and were capable of activating syngeneic CD8+ T cells. In vivo, the rat five-sixths nephrectomy model allowed the localization and activation of renal DC. Accumulation of DC in the renal parenchyma peaked 1 wk after surgery and decreased at 4 wk, concomitant with their appearance in the renal draining lymph nodes. DC from renal lymph nodes, loaded with ALB1-24, activated syngeneic CD8+ T cells in primary culture. The response of CD8+ T cells of five-sixths nephrectomized rats was amplified with secondary stimulation. In contrast, DC from renal lymph nodes of five-sixths nephrectomized rats treated with the proteasomal inhibitor bortezomib lost their capacity to stimulate CD8+ T cells in primary and secondary cultures. These data suggest that albumin can be a source of potentially antigenic peptides upon renal injury and that renal DC play a role in processing self-proteins through a proteasome-dependent pathway.

Reduction of urinary 8‐epi‐prostaglandin F2α during cyclo‐oxygenase inhibition in rats but not in man

1 8‐epi‐prostaglandin (PG) F2α, a major F2 isoprostane, is produced in vivo by free radical‐dependent peroxidation of lipid‐esterified arachidonic acid. Both cyclo‐oxygenase isoforms (COX‐1 and COX‐2) may also form free 8‐epi‐PGF2α as a minor product. It has been recently seen in human volunteers that the overall basal formation of 8‐epi‐PGF2αin vivo is mostly COX‐independent and urinary 8‐epi‐PGF2α is therefore an accurate marker of ‘basal’ oxidative stress in vivo. 2 To test the validity of this marker in the rat, we evaluated in vivo the effect of COX inhibition on the formation of 8‐epi‐PGF2α vs prostanoids. Two structurally unrelated COX inhibitors (naproxen: 30 mg kg−1 day−1; indomethacin: 4 mg kg−1 day−1) were given i.p. to rats kept in metabolic cages. In vivo formation of 8‐epi‐PGF2α was assessed by measuring its urinary excretion. Prostanoid biosynthesis was assessed by measuring urinary excretion of major metabolites of thromboxane (TX) and prostacyclin (2,3‐dinor‐TXB1 and 2,3‐dinor‐6‐keto‐PGF1α). All compounds were selectively measured by immunopurification/gas chromatography‐mass spectrometry. 3 Naproxen reduced urinary excretion of 2,3‐dinor‐TXB1 and 2,3‐dinor‐6‐keto‐PGF1α but, unexpectedly, also that of 8‐epi‐PGF2α (82, 49 and 52% inhibition, respectively). Indomethacin had a similar effect (77, 69 and 55% inhibition). Esterified 8‐epi‐PGF2α in liver and plasma remained unchanged after indomethacin. 4 These findings prompted us to re‐assess the contribution of COX activity to the systemic production of 8‐epi‐PGF2α in man. We gave naproxen (1 g day−1) to healthy subjects (four nonsmokers and four smokers). Urinary 8‐epi‐PGF2α remained unchanged in the two groups (9.63±0.99 before vs 10.24±1.01 after and 20.14±3.00 vs 19.03±2.45 ng h−1 1.73 m−2), whereas there was a marked reduction of major urinary metabolites of thromboxane and prostacyclin (about 90% for both 11‐dehydro‐TXB2 and 2,3‐dinor‐TXB2; >50% for 2,3‐dinor‐6‐keto‐PGF1α). 5 To investigate whether rat COX‐1 produces 8‐epi‐PGF2α more efficiently than human COX‐1, we measured the ex vivo formation of 8‐epi‐PGF2α and TXB2 simultaneously in whole clotting blood. Serum levels of 8‐epi‐PGF2α and TXB2 were similar in rats and man. 6 We conclude that a significant amount of COX‐dependent 8‐epi‐PGF2α is present in rat but not in human urine under normal conditions. This implies that urinary 8‐epi‐PGF2α cannot be used as an index of near‐basal oxidant stress in rats. On the other hand, our data further confirm the validity of this marker in man.

Neuroprotective effects of toll-like receptor 4 antagonism in spinal cord cultures and in a mouse model of motor neuron degeneration.

Sustained inflammatory reactions are common pathological events associated with neuron loss in neurodegenerative diseases. Reported evidence suggests that Toll-like receptor 4 (TLR4) is a key player of neuroinflammation in several neurodegenerative diseases. However, the mechanisms by which TLR4 mediates neurotoxic signals remain poorly understood. We investigated the role of TLR4 in in vitro and in vivosettings of motor neuron degeneration. Using primary cultures from mouse spinal cords, we characterized both the proinflammatory and neurotoxic effects of TLR4 activation with lipopolysaccharide (activation of microglial cells, release of proinflammatory cytokines and motor neuron death) and the protective effects of a cyanobacteriaderived TLR4 antagonist (VB3323). With the use of TLR4-deficient cells, a critical role of the microglial component with functionally active TLR4 emerged in this setting. The in vivo experiments were carried out in a mouse model of spontaneous motor neuron degeneration, the wobbler mouse, where we preliminarily confirmed a protective effect of TLR4 antagonism. Compared with vehicle- and riluzole-treated mice, those chronically treated with VB3323 showed a decrease in microglial activation and morphological alterations of spinal cord neurons and a better performance in the paw abnormality and grip-strength tests. Taken together, our data add new understanding of the role of TLR4 in mediating neurotoxicity in the spinal cord and suggest that TLR4 antagonists could be considered in future studies as candidate protective agents for motor neurons in degenerative diseases.

Effects of cigarette smoking on the human urinary proteome

In this pilot study we used a proteomic approach to compare the urinary protein patterns of healthy smokers and non-smokers. Proteins were resolved by two-dimensional gel electrophoresis and identified by mass spectrometry. The relative abundance of three inflammatory proteins (S100A8, inter-alpha-trypsin inhibitor heavy chain 4, CD59) and that of two isoforms of pancreatic alpha amylase was significantly higher in smokers. Zinc-alpha-2-glycoprotein was the only protein down-regulated in smokers. Its abundance was significantly correlated with urinary glucocorticoids. Most of the proteins identified may be non-specific biomarkers of tobacco effects, since they are involved in inflammatory responses associated with several diseases. Of greater interest are the changes in abundance of pancreatic alpha amylase and zinc-alpha-2-glycoprotein, which after proper validation, might be candidate biomarkers of diseases resulting from exposure to tobacco smoke. The data also show for the first time that smoking can affect the expression profile of urinary proteins.

Secretome Analysis of Multiple Pancreatic Cancer Cell Lines Reveals Perturbations of Key Functional Networks

The cancer secretome is a rich repository in which to mine useful information for both cancer biology and clinical oncology. To help understand the mechanisms underlying the progression of pancreatic cancer, we characterized the secretomes of four human pancreatic ductal adenocarcinoma (PDAC) cell lines versus a normal counterpart. To this end, we used a proteomic workflow based on high-confidence protein identification by mass spectrometry, semiquantitation by a label-free approach, and network enrichment analysis by a system biology tool. Functional networks significantly enriched with PDAC-dysregulated proteins included not only expected alterations within key mechanisms known to be relevant for tumor progression (e.g., cell-cell/cell-matrix adhesion, extracellular matrix remodeling, and cytoskeleton rearrangement), but also other extensive, coordinated perturbations never observed in pancreatic cancer. In particular, we highlighted perturbations possibly favoring tumor progression through immune escape (i.e., inhibition of the complement system, deficiency of selected proteasome components within the antigen-presentation machinery, and inhibition of T cell cytoxicity), and a defective protein folding machinery. Among the proteins found concordantly oversecreted in all of our PDAC cell lines, many are reportedly overexpressed in pancreatic cancer (e.g., CD9 and Vimentin), while others (PLOD3, SH3L3, PCBP1, and SFRS1) represent novel PDAC-secreted proteins that may be worth investigating.

Arachidonic acid metabolic profiles in human meningiomas and gliomas

We determined arachidonic acid (AA) cyclooxygenase metabolic profiles in specimens of human intracranial tumors (gliomas and meningiomas) and, when available, normal brain tissue. Samples were collected at surgery and immediately frozen in liquid nitrogen. The five stable metabolites of AA (PGE2, PGD2, PGF2α, 6-keto-PGF1α and TXB2) were measured by high-resolution gas chromatography-mass spectrometry after ex vivo metabolism of endogenous AA by tissue homogenates. The absolute amounts of AA metabolites varied widely between samples, though meningiomas and gliomas showed characteristic profiles. Compared to the slow-growing benign meningiomas, the rapidly-growing infiltrating gliomas had higher synthesis of TXA2 (reported as a procancer metabolite) and lower synthesis of PGD2 and PGI2 (reported as anticancer metabolites). A higher overall synthesis capacity, preferentially toward TXA2, was found in glioblastomas than in nonpathological brain tissue.