Cocaine in surface waters: a new evidence-based tool to monitor community drug abuse

BACKGROUND: Cocaine use seems to be increasing in some urban areas worldwide, but it is not straightforward to determine the real extent of this phenomenon. Trends in drug abuse are currently estimated indirectly, mainly by large-scale social, medical, and crime statistics that may be biased or too generic. We thus tested a more direct approach based on 'field' evidence of cocaine use by the general population. METHODS: Cocaine and its main urinary metabolite (benzoylecgonine, BE) were measured by mass spectrometry in water samples collected from the River Po and urban waste water treatment plants of medium-size Italian cities. Drug concentration, water flow rate, and population at each site were used to estimate local cocaine consumption. RESULTS: We showed that cocaine and BE are present, and measurable, in surface waters of populated areas. The largest Italian river, the Po, with a five-million people catchment basin, steadily carried the equivalent of about 4 kg cocaine per day. This would imply an average daily use of at least 27 ± 5 doses (100 mg each) for every 1000 young adults, an estimate that greatly exceeds official national figures. Data from waste water treatment plants serving medium-size Italian cities were consistent with this figure. CONCLUSION: This paper shows for the first time that an illicit drug, cocaine, is present in the aquatic environment, namely untreated urban waste water and a major river. We used environmental cocaine levels for estimating collective consumption of the drug, an approach with the unique potential ability to monitor local drug abuse trends in real time, while preserving the anonymity of individuals. The method tested here – in principle extendable to other drugs of abuse – might be further refined to become a standardized, objective tool for monitoring drug abuse

The rapid spread of SARS-COV-2 Omicron variant in Italy reflected early through wastewater surveillance

The SARS-CoV-2 Omicron variant emerged in South Africa in November 2021, and has later been identified worldwide, raising serious concerns. A real-time RT-PCR assay was designed for the rapid screening of the Omicron variant, targeting characteristic mutations of the spike gene. The assay was used to test 737 sewage samples collected throughout Italy (19/21 Regions) between 11 November and 25 December 2021, with the aim of assessing the spread of the Omicron variant in the country. Positive samples were also tested with a real-time RT-PCR developed by the European Commission, Joint Research Centre (JRC), and through nested RT-PCR followed by Sanger sequencing. Overall, 115 samples tested positive for Omicron SARS-CoV-2 variant. The first occurrence was detected on 7 December, in Veneto, North Italy. Later on, the variant spread extremely fast in three weeks, with prevalence of positive wastewater samples rising from 1.0% (1/104 samples) in the week 5–11 December, to 17.5% (25/143 samples) in the week 12–18, to 65.9% (89/135 samples) in the week 19–25, in line with the increase in cases of infection with the Omicron variant observed during December in Italy. Similarly, the number of Regions/Autonomous Provinces in which the variant was detected increased from one in the first week, to 11 in the second, and to 17 in the last one. The presence of the Omicron variant was confirmed by the JRC real-time RT-PCR in 79.1% (91/115) of the positive samples, and by Sanger sequencing in 66% (64/97) of PCR amplicons. In conclusion, we designed an RT-qPCR assay capable to detect the Omicron variant, which can be successfully used for the purpose of wastewater-based epidemiology. We also described the history of the introduction and diffusion of the Omicron variant in the Italian population and territory, confirming the effectiveness of sewage monitoring as a powerful surveillance tool

An Integrated Approach for a Structural and Functional Evaluation of Biosimilars: Implications for Erythropoietin

BACKGROUND: Authorization to market a biosimilar product by the appropriate institutions is expected based on biosimilarity with its originator product. The analogy between the originator and its biosimilar(s) is assessed through safety, purity, and potency analyses. OBJECTIVE: In this study, we proposed a useful quality control system for rapid and economic primary screening of potential biosimilar drugs. For this purpose, chemical and functional characterization of the originator rhEPO alfa and two of its biosimilars was discussed. METHODS: Qualitative and quantitative analyses of the originator rhEPO alfa and its biosimilars were performed using reversed-phase high-performance liquid chromatography (RP-HPLC). The identification of proteins and the separation of isoforms were studied using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF–MS) and two-dimensional gel electrophoresis (2D-PAGE), respectively. Furthermore, the biological activity of these drugs was measured both in vitro, evaluating the TF-1 cell proliferation rate, and in vivo, using the innovative experimental animal model of the zebrafish embryos. RESULTS: Chemical analyses showed that the quantitative concentrations of rhEPO alfa were in agreement with the labeled claims by the corresponding manufacturers. The qualitative analyses performed demonstrated that the three drugs were pure and that they had the same amino acid sequence. Chemical differences were found only at the level of isoforms containing N-glycosylation; however, functional in vitro and in vivo studies did not show any significant differences from a biosimilar point of view. CONCLUSION: These rapid and economic structural and functional analyses were effective in the evaluation of the biosimilarity between the originator rhEPO alfa and the biosimilars analyzed. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s40259-015-0136-3) contains supplementary material, which is available to authorized users

Tumour-derived high molecular weight M-CSF induces monocyte differentiation into M2- polarized macrophages

Experimental and clinical evidence has highlighted that tumor-associated macrophages (TAM) represent the principal component of the leukocyte infiltrate and are usually associated with tumour growth, progression and metastasis. Macrophage population is generally divided into two distinct subsets: M1 and M2. M1 macrophages act as a first line of defence against pathogens whereas M2 cells participate in wound repair and maintenance of tissue integrity. In the tumour micro-environment TAM interactions with the extracellular matrix, neighboring cells, and soluble stimuli largely influence their gene expression and behavior. To investigate the role of the tumor micro-environment on macrophage differentiation, we cultured freshly isolated human monocytes with pancreatic cancer cell line supernatants, in the absence of exogenous cytokine addition.. In selected cultures, about 50% of the monocytes differentiated after 5 days into macrophages. The phenotype analysis of tumor-conditioned macrophages (TC-macro) demonstrated high expression of the mannose receptor, CD16, CD68 and low levels of MHC class II. TC-macro produced IL-10, IL-6, TNF but not IL-12, even after LPS stimulation. Moreover, TC-macro produced a panel of chemokines including CCL2, CXCL8, CCL17 and CXCL10. The transcriptional profile of TC-macro revealed that several genes in line with an M2 polarization are highly expressed. The nature of the tumor-derived factors inducing macrophage differentiation is currently under investigation; biochemical analysis indicated that the biological activity is excluded from exosomes and have a high molecular weight (>100.000 KDa). IL-3 and IL-6 were not detectable in tumor supernatants whereas M-CSF was present at low levels. By mass spectrometric techniques, we surprisingly found that the tumor-derived M-CSF had peculiar migration patterns which were different from those expected for the common human homodimeric glycosilated protein, suggesting an interesting structural differences for the tumor-secreted isoforms of this primary regulator of mononuclear phagocyte. The characterization of tumor-derived factors inducing macrophage differentiation could better clarify the intricate cross-talk between tumor cells and macrophages and thus might aid in the process of devising novel anti-tumor treatments

Tumour-derived high molecular weight M-CSF induces monocyte differentiation into M2- polarized macrophages

Experimental and clinical evidence has highlighted that tumor-associated macrophages (TAM) represent the principal component of the leukocyte infiltrate and are usually associated with tumour growth, progression and metastasis. Macrophage population is generally divided into two distinct subsets: M1 and M2. M1 macrophages act as a first line of defence against pathogens whereas M2 cells participate in wound repair and maintenance of tissue integrity. In the tumour micro-environment TAM interactions with the extracellular matrix, neighboring cells, and soluble stimuli largely influence their gene expression and behavior. To investigate the role of the tumor micro-environment on macrophage differentiation, we cultured freshly isolated human monocytes with pancreatic cancer cell line supernatants, in the absence of exogenous cytokine addition.. In selected cultures, about 50% of the monocytes differentiated after 5 days into macrophages. The phenotype analysis of tumor-conditioned macrophages (TC-macro) demonstrated high expression of the mannose receptor, CD16, CD68 and low levels of MHC class II. TC-macro produced IL-10, IL-6, TNF but not IL-12, even after LPS stimulation. Moreover, TC-macro produced a panel of chemokines including CCL2, CXCL8, CCL17 and CXCL10. The transcriptional profile of TC-macro revealed that several genes in line with an M2 polarization are highly expressed. The nature of the tumor-derived factors inducing macrophage differentiation is currently under investigation; biochemical analysis indicated that the biological activity is excluded from exosomes and have a high molecular weight (>100.000 KDa). IL-3 and IL-6 were not detectable in tumor supernatants whereas M-CSF was present at low levels. By mass spectrometric techniques, we surprisingly found that the tumor-derived M-CSF had peculiar migration patterns which were different from those expected for the common human homodimeric glycosilated protein, suggesting an interesting structural differences for the tumor-secreted isoforms of this primary regulator of mononuclear phagocyte. The characterization of tumor-derived factors inducing macrophage differentiation could better clarify the intricate cross-talk between tumor cells and macrophages and thus might aid in the process of devising novel anti-tumor treatments

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 (M\u3d5) 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 M\u3d5 differentiation due to secreted M-CSF isoforms, including high molecular mass species. A global gene profiling of tumor-conditioned M\u3d5 was performed. Comparison with other datasets (polarized M1-M\u3d5, M2-M\u3d5, and TAMs isolated from human tumors) highlighted the upregulation of several genes also shared by TAM and M2-polarized M\u3d5. 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 M\u3d5. MSF production was confirmed by immunohistochemistry in human TAMs. MSF was induced by M-CSF, IL-4, and TGF\u3b2 but not by proinflammatory stimuli. RNA and protein analysis clearly demonstrated that it is specifically associated with the M2 polarization of M\u3d5. Tumor-conditioned M\u3d5-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 M\u3d5 and expressed by TAMs. Its biological function may contribute to M\u3d5-mediated promotion of cancer cell invasion and metastasis

Differential role of Interleukin-1 and Interleukin-6 in K-Ras-driven pancreatic carcinoma undergoing mesenchymal transition.

K-Ras mutations are a hallmark of human pancreatic adenocarcinoma (PDAC) and epithelial-mesenchymal-transition (EMT) is a driver of progression. Oncogenic K-Ras causes the constitutive activation of NF-kB and the switch-on of an inflammatory program, which further fuels NF-kB and STAT3 activation. In this study we investigated how inflammatory pathways triggered by oncogenic K-Ras are regulated in human pancreatic cancer cells with distict epithelial or mesenchymal phenotype. Our results demonstrate that in cells with epithelial features, K-Ras driven inflammation is under the control of IL-1, while in cells undergoing EMT, is IL-1 independent. In pancreatic tumor cells with EMT phenotype, treatment with IL-1R antagonist (Anakinra) did not inhibit inflammatory cytokine production and tumor growth in mice. In these cells IL-6 is actively transcribed by the EMT transcription factor TWIST. Targeting of mesenchymal pancreatic tumors in vivo with anti-IL-6RmAb (RoActemra) successfully decreased tumor growth in immunodeficient mice, inhibited the inflammatory stroma and NF-kB-p65 and STAT3 phosphorylation in cancer cells. The results confirm that IL-1 is an important driver of inflammation in epithelial pancreatic tumors; however, tumor cells undergoing EMT will likely escape IL-1R inhibition, as IL-6 is continuously transcribed by TWIST. These findings have implications for the rational targeting of inflammatory pathways in human pancreatic cancer