Due to poor prognosis of glioblastoma (GBM), there is an urgent need to develop new therapeutic strategies. Besides eliminating GBM tumor cells and stem cells, a novel therapeutic approach aims to target Glioma-associated microglia/macrophages (GAMs). We investigated the molecular profile of GAMs correlated with patient prognosis by exploiting M1/M2-like polarization markers in a cohort of 20 GBM patients. Using quantitative PCR (qPCR), the markers CXCL10 (M1) and CCL13 (M2) were validated in human macrophages and applied to a global analysis of GBM tissue. Furthermore, proteinase genes, known to be associated with GBM progression (ADAM8, MMP9, MMP14, ADAM10, ADAM17), were analyzed in correlation to M1/M2 markers. Notably, expression levels of ADAM10 and ADAM17 are significantly correlated with an M1-like phenotype and are positively associated to patient survival. Whilst ADAM8 mRNA expression was equally correlated with M1- and M2-like markers, genes for MMP9 and MMP14 are significantly associated with an M2-like phenotype and association to impaired prognosis in the GBM patient cohort. Thus, we provide a robust and reliable combination of qPCR markers to characterize global microglia/macrophage status and the associated proteinase profiles in GBM patients that can be used to analyze the tumor microenvironment, the patients’ prognosis and preselect those GBM patients for which targeting the microglia/macrophage population by repolarization might be beneficial.
We implement dual-volume centrifugal step emulsification on a single chip to extend the dynamic range of digital assays. Compared to published single-volume approaches, the range between the lower detection limit (LDL) and the upper limit of quantification (ULQ) increases by two orders of magnitude. In comparison to existing multivolume approaches, the dual-volume centrifugal step emulsification requires neither complex manufacturing nor specialized equipment. Sample metering into two subvolumes, droplet generation, and alignment of the droplets in two separate monolayers are performed automatically by microfluidic design. Digital quantification is demonstrated by exemplary droplet digital loop-mediated isothermal amplification (ddLAMP). Within 5 min, the reaction mix is split into subvolumes of 10.5 and 2.5 μL, and 2,5k and 176k droplets are generated with diameters of 31.6 ± 1.4 and 213.9 ± 7.5 μm, respectively. After 30 min of incubation, quantification over 5 log steps is demonstrated with a linearity of R 2 ≥ 0.992.
IntroductionYaws, caused by the bacterium Treponema pallidum subsp. pertenue, is a neglected tropical disease targeted for eradication by 2030. Improved diagnostics will be essential to meet this goal. Diagnosis of yaws has relied heavily on clinical and serological tools. However, the presence of coendemic cutaneous skin ulcer diseases, such as lesions caused by Haemophilus ducreyi (HD), means these techniques do not provide a reliable diagnosis. Thus, new diagnostic tools are needed. Molecular tools such as PCR are ideal, but often expensive as they require trained technicians and laboratory facilities, which are often not available to national yaws programmes.Methods and analysisThe LAMP4yaws project is a cross-sectional, observational, diagnostic accuracy study of a combined Treponema pallidum (TP) and HD loop mediated isothermal amplification (TPHD-LAMP) test performed under real world conditions in three endemic countries in West Africa. Individuals with serologically confirmed yaws will be recruited in Cameroon, Côte d’Ivoire and Ghana. Each participant will provide paired swabs, one of which will be sent to the respective national reference laboratory for yaws quantitative PCR and the other will be tested for both TP and HD using the TPHD-LAMP test at local district laboratories. Sensitivity and specificity of the TPHD-LAMP test will be calculated against the reference standard qPCR. We will also assess the acceptability, feasibility and cost-effectiveness of the test. We anticipate that results from this study will support the adoption of the TPHD-LAMP test for use in global yaws eradication efforts.Ethics and disseminationWe have received ethical approval from all relevant institutional and national ethical committees. All participants, or their parents or guardians, must provide written informed consent prior to study enrolment. Study results will be published in an open access journal and disseminated with partners and the World Health Organization.Trial registration numberNCT04753788.
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