Prevalence of HCV among people who inject drugs in Brussels-a respondent-driven sampling survey.

BACKGROUND: In Belgium, people who inject drugs (PWID) are at a high risk of being infected by hepatitis C (HCV) as injecting drug use is the main mode for transmission of HCV in Europe. Estimates about the number of people living with HCV in Belgium are rare and even less is known about the prevalence of HCV among PWID. METHOD: Between 1 February 2019 and 26 April 2019, PWID and high-risk opiate users (HROU) were recruited in Brussels through respondent-driven sampling (RDS). They were invited to a questionnaire and underwent a rapid HCV test. RESULTS: A total of 253 respondents participated in the study, of which 168 were PWID and 238 were HROU, with 153 respondents belonging to both categories. The overall unweighted sample average for HCV antibodies was 41.1%. The weighted population estimates were 43.7% (95% CI 30.6-56.8%) for RDS-II and 43.4% (95% CI 28.9-58.0%) for RDS-SS. CONCLUSIONS: This prevalence is lower than the prevalence estimates reported elsewhere in Europe. However, the data still suggest that serious efforts are needed to reach the goal set by the WHO to reduce HCV by 2030 with 90%.

Awareness of users and motivational factors for using new psychoactive substances in Belgium.

BACKGROUND: Few data on motivations for using new psychoactive substances (NPS) are available. However, the cost, the legal status, and their accessibility through channels like internet contributed to the popularity of NPS. The objective of this article are first to gain a deeper understanding of the culture surrounding NPS in Belgium and second to define the awareness of the users concerning the content of the NPS they are consuming. METHODS: Snowball sampling and partners in the drug demand reduction field were used as a gateway in order to reach a heterogeneous study population. In total, 45 users were recruited and in-depth interviews were conducted. The personal experiences of NPS users and their needs for support along the continuum of care were explored through an interview guideline, while subjects were given the opportunity to deposit a NPS sample for forensic analysis in a recognized laboratory. RESULTS: A diversity of profiles was found among NPS users but also a wide diversity in the motives to consume NPS: personal reasons such as pleasure, mind exploration, being connected to others, or out of curiosity, but also external reasons such as price, accessibility or the specific effects procured by certain NPS. The results showed as well that a majority of NPS users seem to be aware of the substances they are using. CONCLUSION: Understanding the motivations of use is of importance to determine which type of NPS targeted interventions are adapted to different profiles of users.

Laminin-332 sustains chemoresistance and quiescence as part of the human hepatic cancer stem cell niche.

BACKGROUND & AIMS: Cancer stem cells (CSCs) are thought to be persistent in tumours due to their chemoresistance and to cause relapse and metastasis. Hepatic carcinomas displaying hepatic progenitor cell (HPC) features have been associated with a poor prognosis, though it remains unclear how CSCs relate to these different histological subtypes. METHODS: Candidate CSCs were isolated using the side population (SP) technique from primary tissue samples diagnosed as keratin(K)19-negative or -positive hepatocellular carcinoma (HCC) or as combined hepatocellular/cholangiocarcinoma and analysed for gene and protein expression. The effect of laminin-332 was analysed in vitro by using HCC cell lines and in vivo using a xenograft mouse model. RESULTS: The size of the SP correlated with the degree of HPC features found in human hepatic cancer, and also showed an elevated mRNA expression of biliary/HPC markers and the extracellular matrix marker LAMC2, the gene encoding the laminin γ2-chain. Immunopositivity for the γ2-chain of laminin-332 was seen in the extracellular matrix surrounding small HPC-like tumour cells with a low proliferation rate. In vitro, laminin-332 increased K19 expression, phosphorylated mTOR and decreased phospho-histone H3 expression, indicating reduced cell mitosis. The effect of laminin-332 was enhanced upon mTORC1 inhibition and diminished when inhibiting mTORC1+C2. Resistance to doxorubicin and sorafenib treatment, and the SP fraction increased in the coated condition. In vivo, laminin-332 reduced tumour growth and sustained K19 expression. CONCLUSIONS: In this study we identified a prominent role for laminin-332 as part of the specialised CSC niche in maintaining and supporting cell 'stemness', which leads to chemoresistance and quiescence.

Evaluation of an electrochemical sensor and comparison with spectroscopic approaches as used today in practice for harm reduction in a festival setting-A case study: Analysis of 3,4-methylenedioxymethamphetamine samples.

More and more countries and organisations emphasise the value of harm reduction measures in the context of illicit drug use and abuse. One of these measures is drug checking, a preventive action that can represent a quick win by tailored consultation on the risks of substance use upon analytical screening of a submitted sample. Unlike drop-in centres that operate within a fixed setting, enabling drug checking in a harm reduction context at events requires portable, easy to use analytical approaches, operated by personnel with limited knowledge of analytical chemistry. In this case study, four different approaches were compared for the characterisation of 3,4-methylenedioxymethamphetamine samples and this in the way the approaches would be applied today in an event context. The four approaches are mid-infrared (MIR), near-infrared, and Raman spectroscopy, which are today used in drug checking context in Belgium, as well as an electrochemical sensor approach initially developed in the context of law enforcement at ports. The MIR and the electrochemical approach came out best, with the latter allowing for a direct straightforward analysis of the percentage 3,4-methylenedioxymethamphetamine (as base equivalent) in the samples. However, MIR has the advantage that, in a broader drug checking context, it allows to screen for several molecules and so is able to identify unexpected active components or at least the group to which such components belong. The latter is also an important advantage in the context of the growing emergence of new psychotropic substances.

Patient Reported Outcome and Experience Measures (PROMs and PREMs) in substance use disorder treatment services: A scoping review.

BACKGROUND: Substance use disorders (SUD) pose significant challenges for healthcare systems, and there is a need to monitor the provision of effective, individualized care to persons accessing treatment. Patient-Reported Outcome Measures (PROMs) and Patient-Reported Experience Measures (PREMs) are increasingly used in healthcare services to measure treatment outcomes and quality of care as perceived by patients, and to guide service improvement. OBJECTIVES: This review aims to identify and characterize international developments regarding the use and systematic implementation of PROMs and PREMs in SUD treatment services. METHODS: A scoping review was conducted searching multiple databases to identify studies on the use and routine implementation of PROMs and PREMs in SUD treatment services. RESULTS: 23 articles were selected, all dating from 2016 onwards. There was large variation in the patient-reported measures that were used, how they were developed and how and when patient-reported data were collected. Treatment providers identified leadership support, the presence of an integrated electronic patient record, and regular feedback to be the most important facilitators of successful implementation of patient-reported measures into clinical practice, whilst treatment dropout and burden to staff and patients were the most important barriers to consider. CONCLUSIONS: PROMs and PREMs are increasingly used in SUD treatment services, but guidance is needed to support researchers and clinicians in selecting and implementing valid, meaningful, and comparable measures if we want to understand the effects of PROM and PREM data collection and feedback on treatment quality and results.

Human pancreatic adenocarcinoma contains a side population resistant to gemcitabine.

BACKGROUND: Therapy resistance remains one of the major challenges to improve the prognosis of patients with pancreatic cancer. Chemoresistant cells, which potentially also display cancer stem cell (CSC) characteristics, can be isolated using the side population (SP) technique. Our aim was to search for a SP in human pancreatic ductal adenocarcinoma (PDAC) and to examine its chemoresistance and CSC(-like) phenotype. METHODS: Human PDAC samples were expanded in immunodeficient mice and first-generation xenografts analyzed for the presence of a Hoechst dye-effluxing SP using flow cytometry (FACS). To investigate chemoresistance of the SP, mice bearing PDAC xenografts were treated with gemcitabine and SP proportion determined. In addition, the SP and the main tumour cell population (MP) were sorted by FACS for RNA extraction to profile gene expression, and for culturing under sphere-forming conditions. RESULTS: A SP was identified in all PDAC samples, analyzed. This SP was more resistant to gemcitabine than the other tumour cells as examined in vivo. Whole-genome expression profiling of the SP revealed upregulation of genes related to therapy resistance, apoptotic regulation and epithelial-mesenchymal transition. In addition, the SP displayed higher tumourigenic (CSC) activity than the MP as analyzed in vitro by sphere-forming capacity. CONCLUSION: We identified a SP in human PDAC and uncovered a chemoresistant and CSC-associated phenotype. This SP may represent a new therapeutic target in pancreatic cancer. TRIAL REGISTRATION: Clinicaltrials.gov NCT00936104.

Increased glucocorticoid receptor expression and activity mediate the LPS resistance of SPRET/EI mice.

SPRET/Ei mice are extremely resistant to acute LPS-induced lethal inflammation when compared with C57BL/6. We found that in vivo SPRET/Ei mice exhibit strongly reduced expression levels of cytokines and chemokines. To investigate the role of the potent anti-inflammatory glucocorticoid receptor (GR) in the SPRET/Ei phenotype, mice were treated with the GR antagonist RU486 or bilateral adrenalectomy. Under such conditions, both C57BL/6 and SPRET/Ei mice were strongly sensitized to LPS, and the differences in LPS response between SPRET/Ei and C57BL/6 mice were completely gone. These results underscore the central role of GR in the LPS hyporesponsiveness of SPRET/Ei mice. Compared with C57BL/6, SPRET/Ei mice were found to express higher GR levels, which were reflected in increased GR transactivation. Using a backcross mapping strategy, we demonstrate that the high GR transcription levels are linked to the Nr3c1 (GR) locus on chromosome 18 itself. Unexpectedly, SPRET/Ei mice exhibit a basal overactivation of the hypothalamic-pituitary-adrenal axis, namely strongly increased corticosterone levels, ACTH levels, and adrenocortical size. As a consequence of the excess of circulating glucocorticoids (GCs), levels of hepatic gluconeogenic enzymes are increased, and insulin secretion from pancreatic ß-cells is impaired, both of which result in hyperglycemia and glucose intolerance in SPRET/Ei mice. We conclude that SPRET/Ei mice are unique as they display an unusual combination of elevated GR expression and increased endogenous GC levels. Hence, these mice provide a new and powerful tool for the study of GR- and GC-mediated mechanisms, including immune repressive functions, neuroendocrine regulation, insulin secretion, and carbohydrate metabolism.

Use of health care services by people with substance use disorders in Belgium: a register-based cohort study.

BACKGROUND: The objective of the study was to describe the frequencies of health-care utilization by people with substance use disorder (SUD), including contacts with general practitioners (GP), psychiatrists, emergency departments (ED) and hospital admissions and to compare this frequency to the general population. METHODS: Data from the national register of people who were in treatment for SUD between 2011 and 2014 was linked to health care data from the Belgian health insurance (N = 30,905). Four comparators were matched on age, sex and place of residence to each subject in treatment for SUD (N = 123,620). Cases were further divided in five mutually exclusive categories based on the main SUD (opiates, crack/cocaine, stimulants, cannabis and alcohol). We calculated the average number of contacts with GP, psychiatrists and ED, and hospital admissions per person over a ten year period (2008-2017), computed descriptive statistics for each of the SUD and used negative binomial regression models to compare cases and comparators. RESULTS: Over the ten-year period, people in treatment for SUD overall had on average 60 GP contacts, 3.9 psychiatrist contacts, 7.8 visits to the ED, and 16 hospital admissions. Rate ratios, comparing cases and corresponding comparators, showed that people in treatment for SUD had on average 1.9 more contacts with a GP (95 % CI 1.9-2.0), 7.4 more contacts with a psychiatrist (95 % CI 7.0-7.7), 4.2 more ED visits (95 % CI 4.2-4.3), and 6.4 more hospital admissions (95 % CI 6.3-6.5). CONCLUSIONS: The use of health services for people with SUD is between almost two (GP) and seven times (psychiatrist) higher than for comparators. People in treatment for alcohol use disorders use health care services more frequently than people in treatment for other SUD. The use of health services remained stable in the five years before and after the moment people with SUD entered into treatment for SUD. The higher use of primary health care services by people with SUD might indicate that they have higher health care needs than comparators.

Women's views on barriers and facilitators for seeking alcohol and drug treatment in Belgium.

Aims: Although treatment barriers are different for men and women, research is dominated by males' and practitioners' perspectives rather than women's voices. The purpose of this study in Belgium was to identify and obtain a better understanding of the barriers and facilitators for seeking treatment as experienced by substance (ab)using women themselves. Methods: In-depth interviews were conducted with 60 female substance users who utilise(d) outpatient and/or residential treatment services. A content analysis was performed on women's personal accounts of previous treatment experiences as well as their experiences with services along the continuum of care, resulting in practical implications for the organisation of services. Results: Female substance users experience various overlapping - and at times competing - barriers and facilitators when seeking treatment and utilising services. For most women, the threat of losing custody of their children is an essential barrier to treatment, whereas for a significant part of the participants it serves as a motivation to seek help. Also, women report social stigma in private as well as professional contexts as a barrier to treatment. Women further ask for a holistic approach to treatment, which stimulates the healing process of body, mind and spirit, and emphasise the importance of feeling safe in treatment. Participants suggested several changes that could encourage treatment utilisation. Conclusion: Our findings demonstrate the need for a gender-sensitive approach within alcohol and drug services that meets the needs of female substance users, as well as gender-sensitivity within prevention and awareness-raising campaigns, reducing the stigma and facilitating knowledge and awareness among women and society.

Estimates of people who injected drugs within the last 12 months in Belgium based on a capture-recapture and multiplier method.

BACKGROUND: For Belgium, available estimates of the number of people who inject drugs (PWID) are based on data from more than fifteen years ago and apply only to those who report ever injecting drugs. As a result, no reliable baseline data exist to determine the scale of services for PWID. METHODS: We obtained pseudo-anonymized identifier information from treatment and harm reduction service providers and a fieldwork study between February and April 2019 in Brussels. We estimated the number of PWID, defined as people who injected within the last 12 months, in Brussels using capture-recapture (CRC) methodology. To obtain national estimates, we scaled the proportion of PWID in Brussels to the total number of this population in Belgium based on two existing drug treatment registers, which were then multiplied with the result of the CRC. RESULTS: The total population of PWID is estimated to be 703 (95 %CI 538-935) for Brussels and between 6620 (95 %CI 4711 - 8576) and 7018 (95 %CI 4794 - 9527) for Belgium. CONCLUSIONS: These estimates provide crucial information to ensure that services to PWID are adequately maintained. They clearly indicate the need to maximize efforts to achieve the targets set by WHO for 2030 on the provision of 300 sterile needles and syringes per PWID per year, a 90 % reduction of new HCV infections, and a 65 % reduction of liver-related mortality.

COVID-19 and people who use drugs: Impact of the pandemic on general anxiety and depressive disorders among adults in Belgium.

BACKGROUND: During previous pandemics people who use drugs (PWUD) were categorized among the most vulnerable. In the current study, firstly, we wanted to evaluate the impact of the COVID-19 crisis on the prevalence of anxiety and depressive disorders among PWUD. Furthermore, we wanted to compare the prevalence of these disorders with that of members from the general population who did not use drugs. METHODS: We used a matched cohort design based on two separate repeated cross-sectional online surveys (April and November 2020) among PWUD and the general population. Results of GAD-7 and PHQ-9 were used as outcome variables. We calculated absolute and relative risks for matched pairs for both affective disorders, and logistic regression to compare affective disorders over both waves for PWUD. RESULTS: In April, the prevalence of affective disorders was similar for PWUD and the general population. In November, the risks for anxiety disorders increased with 64% for PWUD compared to non-PWUD (RR = 1.64, 95%CI 1.42-1.88), whereas the risks for depressive disorders more than doubled (RR = 2.29, 95%CI 1.97-2.67). Having a job and being male were protective factors for PWUD for both anxiety and depressive disorders. LIMITATIONS: As this study used self-reported data, GAD-7 and PHQ-9 give an indication of the presence of anxiety and depression which might differ from a clinician's judgement. CONCLUSIONS: PWUD might be disproportionally affected by COVID-19. Health care providers should be attentive to substance use as an indicator for increased risk of mental health problems.

Pituitary progenitor cells tracked down by side population dissection.

The pituitary gland represents the endocrine core, governing the body's hormonal landscape by adapting its cellular composition to changing demands. It is assumed that stem/progenitor cells are involved in this remodeling. Recently, we uncovered a candidate stem/progenitor cell population in the anterior pituitary. Here, we scrutinized this "side population" (SP) and show that, unexpectedly, not the subset expressing high levels of "stem cell antigen-1" (Sca1(high)) but the remainder non-Sca1(high) fraction clusters the pituitary progenitor cells. Transcriptomal interrogation revealed in the non-Sca1(high) SP upregulated expression of the pituitary stem/progenitor cell markers Sox2 and Sox9, and of multiple factors critically involved in pituitary embryogenesis. The non-Sca1(high) SP encloses the cells that generate spheres and display multipotent hormone differentiation capacity. In culture conditions selecting for the non-Sca1(high) subset within the SP, stem cell growth factors that induce SP expansion, affect transcription of embryonic factors, suggesting impact on a developmental program that unfolds within this SP compartment. Non-Sca1(high) SP cells, revealed by Sox2 expression, are observed in the postulated periluminal stem/progenitor cell niche, but also in small groups scattered over the gland, thereby advocating the existence of multiple niches. In early postnatal mice undergoing a pituitary growth wave, Sox2(+) cells are more abundant than in adults, concordant with a larger SP and higher non-Sca1(high) proportion. Together, we tracked down pituitary progenitor cells by SP phenotype, and thus provide a straightforward method to isolate and scrutinize these cells from the plastic pituitary ex vivo, as well as a culture system for in-depth exploration of their regulatory network.

Stem cells in the pituitary gland: A burgeoning field.

The pituitary gland represents the endocrine core of the organism, and is well-known for its cellular plasticity in order to meet the body's fluctuating hormonal demands. In the past, it has repeatedly been postulated that the pituitary harbors tissue-specific stem cells that participate in the generation of new endocrine cells during this dynamic cell remodeling, as well as during the slow but robust homeostatic turnover of the gland. However, their presence and identity remained elusive until this conundrum recently attracted renewed interest. Our discovery of a 'side population' using flow cytometry was the first step towards a more convincing candidate stem/progenitor cell population in the endocrine anterior pituitary. Since then, several other groups have endeavored to search for pituitary stem/progenitor cells, which finally culminated in the identification of very strong candidates. Multiple markers were put forward, among which the pluripotency transcription factor Sox2 occupies center stage. Now that very plausible pituitary stem/progenitor cells can be isolated and assayed, detailed characterization of their involvement in pituitary cell remodeling during basal renewal, dynamic adaptation and potential response to injury is around the corner, and is expected to significantly advance our knowledge on pituitary biology. In addition, a comprehensive study of the stem/progenitor cells may guide us to a better understanding of pituitary hormonal deficiencies, as well as of pituitary tumorigenesis in which 'cancer stem cells' may play a central role. Nevertheless, many questions remain to be resolved, and future challenges are huge. In this review, we provide a detailed overview of the exciting recent developments in the pituitary stem cell quest. In addition we pinpoint some discordant findings and include a number of cautionary and critical reflections. The recent acceleration in pituitary stem cell research may initiate a very exciting era in the pituitary field. May we say that "La nouvelle hypofyse est arrivée"?

Alternatives to prison for drug offenders in Belgium during the past decade.

OBJECTIVE: Imprisonment has a more pronounced criminogenic effect on drug offenders than on other types of offenders. Additionally, little research has been conducted on the practical application of drug-related alternatives to prison. Therefore, this study describes drug-related alternatives to prison in Belgium over a ten years' period since 2005. METHODS: The applied drug-related alternatives to prison ('probation', 'conditional release', 'mediation in criminal cases', 'community service' and 'electronic monitoring') were subject to a secondary data analysis of the database of the Houses of Justice. RESULTS: Men, the age group of 16-24 years old and Belgians are most sentenced to alternatives to prison. Nevertheless, 38% of women are guided towards 'probation' while 8% is 'mediated in criminal cases' compared to 30% and 5% of males respectively (p < .001). 26% of non-Belgians are involved in 'conditional release' and 'electronic monitoring' compared to 22% and 16% among Belgians (p < .001) respectively. With regards to age, 21% of the offenders older than 24 years are involved in 'electronic monitoring' compared to 6% among the offenders younger than 25 years (p < .001). CONCLUSIONS: The results highlight differences in punishment judgments by age, gender and nationality that continues to be indicative for perceived threat, danger and culpability of the offenders.

Utilization of gabapentin by people in treatment for substance use disorders in Belgium (2011-2014): a cross-sectional study.

BACKGROUND: Although gabapentin has been licensed in the European Union only for neuropathic pain and epilepsy for patients who have partial seizures, it has also been prescribed in treatment for substance use disorders. Many studies report the potential risk of abuse of gabapentin by people with substance use disorders. The objective of this paper is to determine if people who have been in treatment for substance use disorders bought gabapentin in a time span that could indicate consumption at a dose that exceeded the maximum approved dose of 3600 mg/day. METHODS: This analysis is the result of an observational cross-sectional descriptive study with matching. Two datasets were used and linked at individual level. Subjects were selected based on their first registration in the database of the Treatment Demand Indicator (TDI) between 2011 and 2014, without any exclusion criteria concerning nationality or age. Through linkage with the database of the InterMutualistic Agency (IMA) information on health service use and medication use was determined. In addition, each subject was matched on age, sex and place of residence to four comparators from the general population who were not in specialized treatment. The prevalence of gabapentin purchases in the period between 2008 and 2014 for both populations were compared. Quantification of the amount of gabapentin between two consecutive purchases was used as a proxy for potential abuse. RESULTS: Out of 30,905 patients in treatment for substance use disorders 2.7% had bought at least once gabapentin in a public pharmacy or received it from a hospital pharmacy, compared to 0.7% in the comparison group (n = 122,142). In both populations, more than half of the patients bought only once or twice gabapentin and about 10.0% bought at least once gabapentin in a time span that could indicate potential abuse. A limitation of the study is that it is only based on reimbursed medication without clinical information. CONCLUSION: Through the linkage of the TDI-database and the database of the Belgian health insurance companies, no evidence was found for regular abuse of prescribed gabapentin in Belgium by people in treatment for substance use disorders.

Longitudinal pharmacoepidemiological and health services research for substance users in treatment: protocol of the Belgian TDI-IMA linkage.

BACKGROUND: Not much is known about the health seeking behavior of people with substance use disorders before they enter specialized treatment and afterwards. This paper explains in detail the protocol that has been followed to establish the Belgian TDI-IMA-database, which is linking two separate databases: the Treatment Demand Indicator (TDI) and the database of the Intermutualistic Agency (IMA). The Treatment Demand Indicator is measuring incidence of people with substance use disorders entering drug treatment. The IMA-database covers data, collected in the framework of the compulsory Belgian health care and benefits insurance program, on reimbursed medication and the use of reimbursed health services. The linkage results in pharmacoepidemiological and health service data for people who were in treatment for substance use disorders and for a group of comparators. METHODS: The TDI-database was linked to the IMA-database for the period between 01/01/2008 and 31/12/2017, based on the national identification number of patients who have been in alcohol or drug treatment between 01/01/2011 and 31/12/2014. Through this linkage, pharmacoepidemiological and health service data became available for at least 3 years before the first registered episode in the TDI-database till at least 3 years after the first episode. For each person in TDI four comparators, who were not in specialized treatment, were matched on age, sex and place of residence. DISCUSSION: The TDI-IMA-database allows for an analysis of health seeking behavior and health care pathways of people before and after they entered specialized alcohol and drug treatment. The presented protocol could be used in other European countries to establish a linkage between existing health databases. This will allow for a better understanding of the health care needs of patients with substance use disorders.

Treatment for substance use disorders: the Belgian Treatment Demand Indicator registration protocol.

BACKGROUND: Registration of patients with substance use disorders is of key importance to get insights and to study trends in patients characteristics and substance use patterns. The Treatment Demand Indicator (TDI) is gathering this information at European level since 2000. In Belgium, this registration started at national level in 2011 and an increasing number of facilities of different types are participating in this data collection since then. METHODS/DESIGN: This surveillance register collects information on every treatment episode started by patients for their substance use disorder. Information is collected on socio-demographic characteristics of the patient, treatment history and substance use patterns. Patients are identified uniquely using their national identification number in order identify multiple episodes followed by a same person. A large range of treatment facilities have the possibility to participate in this registration to allow a wide coverage of the population. DISCUSSION: The objective of the paper is to facilitate the use of data by authorities or researchers by correctly describing all aspects of the indicator. The case definition, the variables collected and the way data should be reported are of key importance to use and interpret the data correctly. An overview of the data registered in 2014 gives also an idea of the content of the database. The article also pictures the strengths and limitations of the register and foresees some future improvements.

Qualitative screening of new psychoactive substances in pooled urine samples from Belgium and United Kingdom.

Concerns about new psychoactive substances (NPS) are increasing due to the rising frequency of serious intoxications. Analysis of biological fluids (urine) is necessary to get reliable information about the use of these substances. However, it is a challenging task due to the lack of analytical standards and the dynamic character of the NPS market. In the present work, a qualitative screening of NPS was carried out in 23 pooled urine samples collected from a city center in the UK and festivals in the UK and Belgium. The analytical method was based on data-independent acquisition mode using liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. An in-house library was used with >1500 entries corresponding to NPS, classical drugs and metabolites. All samples contained 53 and 28 compounds of interest from the UK and Belgium respectively. Of the different compounds detected, about 70% were confirmed using retention time and product ions while the remaining compounds were identified using elucidated fragmentation pathways. The highest numbers of NPS identified in both countries were from the cathinone and phenylethylamine families, with a higher number being detected in samples from the festival in the UK. Moreover, several cathinone metabolites in human urine were detected and identified. The screening method proved useful to detect a large number of compounds and determine the use of NPS.

Pituitary tumors contain a side population with tumor stem cell-associated characteristics.

Pituitary adenomas cause significant endocrine and mass-related morbidity. Little is known about the mechanisms that underlie pituitary tumor pathogenesis. In the present study, we searched for a side population (SP) in pituitary tumors representing cells with high efflux capacity and potentially enriched for tumor stem cells (TSCs). Human pituitary adenomas contain a SP irrespective of hormonal phenotype. This adenoma SP, as well as the purified SP (pSP) that is depleted from endothelial and immune cells, is enriched for cells that express 'tumor stemness' markers and signaling pathways, including epithelial-mesenchymal transition (EMT)-linked factors. Pituitary adenomas were found to contain self-renewing sphere-forming cells, considered to be a property of TSCs. These sphere-initiating cells were recovered in the pSP. Because benign pituitary adenomas do not grow in vitro and have failed to expand in immunodeficient mice, the pituitary tumor cell line AtT20 was further used. We identified a SP in this cell line and found it to be more tumorigenic than the non-SP 'main population'. Of the two EMT regulatory pathways tested, the inhibition of chemokine (C-X-C motif) receptor 4 (CXCR4) signaling reduced EMT-associated cell motility in vitro as well as xenograft tumor growth, whereas the activation of TGFß had no effect. The human adenoma pSP also showed upregulated expression of the pituitary stem cell marker SOX2. Pituitaries from dopamine receptor D2 knockout (Drd2(-/-)) mice that bear prolactinomas contain more pSP, Sox2(+), and colony-forming cells than WT glands. In conclusion, we detected a SP in pituitary tumors and identified TSC-associated characteristics. The present study adds new elements to the unraveling of pituitary tumor pathogenesis and may lead to the identification of new therapeutic targets.

Human pancreatic cancer contains a side population expressing cancer stem cell-associated and prognostic genes.

In many types of cancers, a side population (SP) has been identified based on high efflux capacity, thereby enriching for chemoresistant cells as well as for candidate cancer stem cells (CSC). Here, we explored whether human pancreatic ductal adenocarcinoma (PDAC) contains a SP, and whether its gene expression profile is associated with chemoresistance, CSC and prognosis. After dispersion into single cells and incubation with Hoechst dye, we analyzed human PDAC resections specimens using flow cytometry (FACS). We identified a SP and main population (MP) in all human PDAC resection specimens (n = 52) analyzed, but detected immune (CD45(+)) and endothelial (CD31(+)) cells in this fraction together with tumor cells. The SP and MP cells, or more purified fractions depleted from CD31(+)/CD45(+) cells (pSP and pMP), were sorted by FACS and subjected to whole-genome expression analysis. This revealed upregulation of genes associated with therapy resistance and of markers identified before in putative pancreatic CSC. pSP gene signatures of 32 or 10 up- or downregulated genes were developed and tested for discriminatory competence between pSP and pMP in different sets of PDAC samples. The prognostic value of the pSP genes was validated in a large independent series of PDAC patients (n = 78) using nCounter analysis of expression (in tumor versus surrounding pancreatic tissue) and Cox regression for disease-free and overall survival. Of these genes, expression levels of ABCB1 and CXCR4 were correlated with worse patient survival. Thus, our study for the first time demonstrates that human PDAC contains a SP. This tumor subpopulation may represent a valuable therapeutic target given its chemoresistance- and CSC-associated gene expression characteristics with potential prognostic value.