Cindy Leten

The Capsaicin Receptor TRPV1 Is a Crucial Mediator of the Noxious Effects of Mustard Oil

Mustard oil (MO) is a plant-derived irritant that has been extensively used in experimental models to induce pain and inflammation. The noxious effects of MO are currently ascribed to specific activation of the cation channel TRPA1 in nociceptive neurons. In contrast to this view, we show here that the capsaicin receptor TRPV1 has a surprisingly large contribution to aversive and pain responses and visceral irritation induced by MO. Furthermore, we found that this can be explained by previously unknown properties of this compound. First, MO has a bimodal effect on TRPA1, producing current inhibition at millimolar concentrations. Second, it directly and stably activates mouse and human recombinant TRPV1, as well as TRPV1 channels in mouse sensory neurons. Finally, physiological temperatures enhance MO-induced TRPV1 stimulation. Our results refute the dogma that TRPA1 is the sole nocisensor for MO and motivate a revision of the putative roles of these channels in models of MO-induced pain and inflammation. We propose that TRPV1 has a generalized role in the detection of irritant botanical defensive traits and in the coevolution of multiple mammalian and plant species.

In vivo and ex vivo assessment of the blood brain barrier integrity in different glioblastoma animal models.

Blood brain barrier (BBB) disruption is used (pre)clinically as a measure for brain tumor malignancy and grading. During treatment it is one of the parameters followed rigorously to assess therapeutic efficacy. In animal models, both invasive and non-invasive methods are used to determine BBB disruption, among them Evans blue injection prior to sacrifice and T1-weighted magnetic resonance imaging (MRI) post contrast injection. In this study, we have assessed the BBB integrity with the methods mentioned above in two experimental high grade glioma models, namely the GL261 mouse glioblastoma model and the Hs683 human oligodendroglioma model. The GL261 model showed clear BBB integrity loss with both, contrast-enhanced (CE) MRI and Evans blue staining. In contrast, the Hs683 model only displayed BBB disruption with CE-MRI, which was not evident on Evans blue staining, indicating a limited BBB disruption. These results clearly indicate the importance of assessing the BBB integrity status using appropriate methods. Especially when using large therapeutic molecules that have difficulties crossing the BBB, care should be taken with the appropriate BBB disruption assessment studies.

Controlling and Monitoring Stem Cell Safety In Vivo in an Experimental Rodent Model

Adult stem cells have been investigated increasingly over the past years for multiple applications. Although they have a more favorable safety profile compared to pluripotent stem cells, they are still capable of self‐renewal and differentiate into several cell types. We investigated the behavior of Oct4‐positive (Oct4+) and Oct4‐negative (Oct4−) murine or rat bone marrow (BM)‐derived stem cells in the healthy brain of syngeneic mice and rats. Engraftment of mouse and rat Oct4‐positive BM‐derived hypoblast‐like stem cells (m/rOct4+ BM‐HypoSCs) resulted in yolk‐sac tumor formation in the healthy brain which was monitored longitudinally using magnetic resonance imaging (MRI) and bioluminescence imaging (BLI). Contrast enhanced MRI confirmed the disruption of the blood brain barrier. In contrast, m/r Oct4‐negative BM‐derived multipotent adult progenitor cells (m/rOct4− BM‐MAPCs) did not result in mass formation after engraftment into the brain. mOct4+ BM‐HypoSCs and mOct4− BM‐MAPCs were transduced to express enhanced green fluorescent protein, firefly luciferase (fLuc), and herpes simplex virus‐thymidine kinase to follow up suicide gene expression as a potential “safety switch” for tumor‐forming stem cells by multimodal imaging. Both cell lines were eradicated efficiently in vivo by ganciclovir administration indicating successful suicide gene expression in vivo, as assessed by MRI, BLI, and histology. The use of suicide genes to prevent tumor formation is in particular of interest for therapeutic approaches where stem cells are used as vehicles to deliver therapeutic genes. Stem Cells 2014;32:2833–2844

Monitoring the Bystander Killing Effect of Human Multipotent Stem Cells for Treatment of Malignant Brain Tumors

Tumor infiltrating stem cells have been suggested as a vehicle for the delivery of a suicide gene towards otherwise difficult to treat tumors like glioma. We have used herpes simplex virus thymidine kinase expressing human multipotent adult progenitor cells in two brain tumor models (hU87 and Hs683) in immune-compromised mice. In order to determine the best time point for the administration of the codrug ganciclovir, the stem cell distribution and viability were monitored in vivo using bioluminescence (BLI) and magnetic resonance imaging (MRI). Treatment was assessed by in vivo BLI and MRI of the tumors. We were able to show that suicide gene therapy using HSV-tk expressing stem cells can be followed in vivo by MRI and BLI. This has the advantage that (1) outliers can be detected earlier, (2) GCV treatment can be initiated based on stem cell distribution rather than on empirical time points, and (3) a more thorough follow-up can be provided prior to and after treatment of these animals. In contrast to rodent stem cell and tumor models, treatment success was limited in our model using human cell lines. This was most likely due to the lack of immune components in the immune-compromised rodents.

Assessment of bystander killing-mediated therapy of malignant brain tumors using a multimodal imaging approach

IntroductionIn this study, we planned to assess if adult stem cell-based suicide gene therapy can efficiently eliminate glioblastoma cells in vivo. We investigated the therapeutic potential of mouse Oct4− bone marrow multipotent adult progenitor cells (mOct4− BM-MAPCs) in a mouse glioblastoma model, guided by multimodal in vivo imaging methods to identify therapeutic windows.MethodsMagnetic resonance imaging (MRI) of animals, wherein 5 × 105 syngeneic enhanced green fluorescent protein-firefly luciferase-herpes simplex virus thymidine kinase (eGFP-fLuc-HSV-TK) expressing and superparamagnetic iron oxide nanoparticle labeled (1 % or 10 %) mOct4− BM-MAPCs were grafted in glioblastoma (GL261)-bearing animals, showed that labeled mOct4− BM-MAPCs were located in and in close proximity to the tumor. Subsequently, ganciclovir (GCV) treatment was commenced and the fate of both the MAPCs and the tumor were followed by multimodal imaging (MRI and bioluminescence imaging).ResultsIn the majority of GCV-treated, but not phosphate-buffered saline-treated animals, a significant difference was found in mOct4− BM-MAPC viability and tumor size at the end of treatment. Noteworthy, in some phosphate-buffered saline-treated animals (33 %), a significant decrease in tumor size was seen compared to sham-operated animals, which could potentially also be caused by a synergistic effect of the immune-modulatory stem cells.ConclusionsSuicide gene therapy using mOct4− BM-MAPCs as cellular carriers was effective in reducing the tumor size in the majority of the GCV-treated animals leading to a longer progression-free survival compared to sham-operated animals. This treatment could be followed and guided noninvasively in vivo by MRI and bioluminescence imaging. Noninvasive imaging is of particular interest for a rapid and efficient validation of stem cell-based therapeutic approaches for glioblastoma and hereby contributes to a better understanding and optimization of a promising therapeutic approach for glioblastoma patients.