Functional detection of MDR1/P170 and MRP/P190-mediated multidrug resistance in tumour cells by flow cytometry.

Multidrug resistance (MDR) in tumour cells is often caused by the overexpression of the plasma membrane drug transporter P-glycoprotein (P-gp) or the recently discovered multidrug resistance-associated protein (MRP). In this study we investigated the specificity and sensitivity of the fluorescent probes rhodamine 123 (R123), daunorubicin (DNR) and calcein acetoxymethyl ester (calcein-AM) in order to detect the function of the drug transporters P-gp and MRP, using flow cytometry. The effects of modulators on the accumulation and retention of these probes were compared in several pairs of sensitive and P-gp- as well as MRP-overexpressing cell lines. R123, in combination with the modulator PSC833, provided the most sensitive test for detecting P-gp-mediated resistance. Moreover, in a 60 min drug accumulation assay R123 can be regarded as a P-gp-specific probe, since R123 is not very efficiently effluxed by MRP. In contrast to R123, a 60 min DNR or calcein-AM accumulation test could be used to detect MRP-mediated resistance. The MRP-specific modulator genistein could be used in combination with DNR, but not with calcein-AM. Vincristine (VCR) can be used to increase the cellular uptake of calcein-AM in MDR cells, but is not specific for MRP. Thus, although the combination of DNR with genistein appeared to be as sensitive as the combination of calcein-AM with VCR, the former may be used to probe specific MRP activity whereas the latter provides a combined (P-gp + MRP) functional MDR parameter. With these functional assays the role and relative importance of P-gp and MRP can be studied in, for example, haematological malignancies

Postcolonial Transplants: Cinema, Diaspora and the Body Politic

This essay examines depictions of migrant workers in French and British postcolonial cinema as transplanted interlopers, 'exotic' or transgendered bodies that are perceived as a threat to the integrity of the body politic

A prospective study on rapid exome sequencing as a diagnostic test for multiple congenital anomalies on fetal ultrasound

Objective: Conventional genetic tests (quantitative fluorescent-PCR [QF-PCR] and single nucleotide polymorphism-array) only diagnose ~40% of fetuses showing ultrasound abnormalities. Rapid exome sequencing (rES) may improve this diagnostic yield, but includes challenges such as uncertainties in fetal phenotyping, variant interpretation, incidental unsolicited findings, and rapid turnaround times. In this study, we implemented rES in prenatal care to increase diagnostic yield. Methods: We prospectively studied 55 fetuses. Inclusion criteria were: (a) two or more independent major fetal anomalies, (b) hydrops fetalis or bilateral renal cysts alone, or (c) one major fetal anomaly and a first-degree relative with the same anomaly. In addition to conventional genetic tests, we performed trio rES analysis using a custom virtual gene panel of ~3850 Online Mendelian Inheritance in Man (OMIM) genes. Results: We established a genetic rES-based diagnosis in 8 out of 23 fetuses (35%) without QF-PCR or array abnormalities. Diagnoses included MIRAGE (SAMD9), Zellweger (PEX1), Walker-Warburg (POMGNT1), Noonan (PTNP11), Kabuki (KMT2D), and CHARGE (CHD7) syndrome and two cases of Osteogenesis Imperfecta type 2 (COL1A1). In six cases, rES diagnosis aided perinatal management. The median turnaround time was 14 (range 8-20) days. Conclusion: Implementing rES as a routine test in the prenatal setting is challenging but technically feasible, with a promising diagnostic yield and significant clinical relevance

Do health systems delay the treatment of poor children? A qualitative study of child deaths in rural Tanzania.

Child mortality remains one of the major public-health problems in Tanzania. Delays in receiving and accessing adequate care contribute to these high rates. The literature on public health often focuses on the role of mothers in delaying treatment, suggesting that they contact the health system too late and that they prefer to treat their children at home, a perspective often echoed by health workers. Using the three-delay methodology, this study focus on the third phase of the model, exploring the delays experienced in receiving adequate care when mothers with a sick child contact a health-care facility. The overall objective is to analyse specific structural factors embedded in everyday practices at health facilities in a district in Tanzania which cause delays in the treatment of poor children and to discuss possible changes to institutions and social technologies. The study is based on qualitative fieldwork, including in-depth interviews with sixteen mothers who have lost a child, case studies in which patients were followed through the health system, and observations of more than a hundred consultations at all three levels of the health-care system. Data analysis took the form of thematic analysis. Focusing on the third phase of the three-delay model, four main obstacles have been identified: confusions over payment, inadequate referral systems, the inefficient organization of health services and the culture of communication. These impediments strike the poorest segment of the mothers particularly hard. It is argued that these delaying factors function as 'technologies of social exclusion', as they are embedded in the everyday practices of the health facilities in systematic ways. The interviews, case studies and observations show that it is especially families with low social and cultural capital that experience delays after having contacted the health-care system. Reductions of the various types of uncertainty concerning payment, improved referral practices and improved communication between health staff and patients would reduce some of the delays within health facilities, which might feedback positively into the other two phases of delay

Clinical-grade N-(4-[18F]fluorobenzoyl)-interleukin-2 for PET imaging of activated T-cells in humans

BACKGROUND: Molecular imaging of immune cells might be a potential tool for response prediction, treatment evaluation and patient selection in inflammatory diseases as well as oncology. Targeting interleukin-2 (IL2) receptors on activated T-cells using positron emission tomography (PET) with N-(4-[18F]fluorobenzoyl)-interleukin-2 ([18F]FB-IL2) could be such a strategy. This paper describes the challenging translation of the partly manual labeling of [18F]FB-IL2 for preclinical studies into an automated procedure following Good Manufacturing Practices (GMP), resulting in a radiopharmaceutical suitable for clinical use. METHODS: The preclinical synthesis of [18F]FB-IL2 was the starting point for translation to a clinical production method. To overcome several challenges, major adaptations in the production process were executed. The final analytical methods and production method were validated and documented. All data with regards to the quality and safety of the final drug product were documented in an investigational medicinal product dossier. RESULTS: Restrictions in the [18F]FB-IL2 production were imposed by hardware configuration of the automated synthesis equipment and by use of disposable cassettes. Critical steps in the [18F]FB-IL2 production comprised the purification method, stability of recombinant human IL2 and the final formulation. With the GMP compliant production method, [18F]FB-IL2 could reliably be produced with consistent quality complying to all specifications. CONCLUSIONS: To enable the use of [18F]FB-IL2 in clinical studies, a fully automated GMP compliant production process was developed. [18F]FB-IL2 is now produced consistently for use in clinical studies

Full Circle or Spiralling Out of Control?: State Violence and the Control of Urbanisation in Papua New Guinea

There is an administrative reluctance to recognise the permanency of urban settlement in Papua New Guinea. This reluctance, evident since the 1960s, has been characteristic of both the colonial and post-colonial administrations. Opposition to some facets of urbanisation continues today, despite growing population and land pressures in most rural areas and real problems of landlessness emerging in particular rural areas. Colonial control of urban populations has been replicated in contemporary times, often in more draconian form. Eviction of urban settlers has been tied to issues of crime and urban respectability, and lingering perceptions that Melanesians should be rural residents. The growth of informal settlements and urbanisation are not seen as issues of urban planning, nor is the context of urban migration linked to socioeconomic inequality, hence other forms of urban policy are largely absent. Strengthening alliances between land-owners and the state (especially police and provincial administrations) have thus emphasised intraurban inequality and hampered national development

Lung resistance-related protein as a predictor of clinical outcome in advanced testicular germ-cell tumours

This study was undertaken to investigate the expression and predictive value for outcome of multidrug resistance-associated (MDR) proteins P-glycoprotein (Pgp), MRP1, BCRP, and LRP, in advanced testicular germ-cell tumours (TGCT). Paraffin-embedded sections from 56 previously untreated patients with metastatic TGCT were immunostained for Pgp, MRP1, BCRP, and LRP. All patients received platinum-based chemotherapy after orchidectomy. Immunostaining was related to clinicopathological parameters, response to chemotherapy, and outcome. Strong and intermediate expressions of the different MDR-related proteins were: 27 and 41% (Pgp), 54 and 37% (MRP1), 86 and 7% (BCRP), and 14 and 29% (LRP). P-glycoprotein and MRP1 associated, respectively, to low AFP (P=0.026) and high LDH levels (P=0.014), whereas LRP expression associated with high beta-hCG levels (P=0.003) and stage IV tumours (P=0.029). No correlation was found between Pgp, MRP1, and BCRP expression and response to chemotherapy and survival. In contrast, patients with LRP-positive tumours (strong or intermediate expression) had shorter progression-free (P=0.0006) and overall survival (P=0.0116) than LRP-negative patients, even after individual log-rank adjustments by statistically associated variables. Our data suggest that a positive LRP immunostaining at the time of diagnosis in metastatic TGCT is associated with an adverse clinical outcome