Anti-inflammatory, but not osteoprotective, effect of the TRAF6/CD40 inhibitor 6877002 in rodent models of local and systemic osteolysis.

NFκB plays a key role in inflammation and skeletal disorders. Previously, we reported that pharmacological inhibition of NFκB at the level of TRAF6 suppressed RANKL, CD40L and IL1ß-induced osteoclastogenesis and attenuated cancer-induced bone disease. TNFα is also known to regulate TRAF6/NFκB signalling, however the anti-inflammatory and osteoprotective effects associated with inhibition of the TNFα/TRAF6/NFκB axis have not been investigated. Here, we show that in vitro and ex vivo exposure to the verified small-molecule inhibitor of TRAF6, 6877002 prevented TNFα-induced NFκB activation, osteoclastogenesis and calvarial osteolysis, but it had no effects on TNFα-induced apoptosis or growth inhibition in osteoblasts. Additionally, 6877002 disrupted T-cells support for osteoclast formation and synoviocyte motility, without affecting the viability of osteoblasts in the presence of T-cells derived factors. Using the collagen-induced arthritis model, we show that oral and intraperitoneal administration of 6877002 in mice reduced joint inflammation and arthritis score. Unexpectedly, no difference in trabecular and cortical bone parameters were detected between vehicle and 6877002 treated mice, indicating lack of osteoprotection by 6877002 in the arthritis model described. Using two independent rodent models of osteolysis, we confirmed that 6877002 had no effect on trabecular and cortical bone loss in both osteoporotic rats or RANKL- treated mice. In contrast, the classic anti-osteolytic alendronate offered complete osteoprotection in RANKL- treated mice. In conclusion, TRAF6 inhibitors may be of value in the management of the inflammatory component of bone disorders, but may not offer protection against local or systemic bone loss, unless combined with anti-resorptive therapy such as bisphosphonates.

Disentangling inflammatory from fibrotic disease activity by fibroblast activation protein imaging.

OBJECTIVES: To date, there is no valuable tool to assess fibrotic disease activity in humans in vivo in a non-invasive way. This study aims to uncouple inflammatory from fibrotic disease activity in fibroinflammatory diseases such as IgG4-related disease. METHODS: In this cross-sectional clinical study, 27 patients with inflammatory, fibrotic and overlapping manifestations of IgG4-related disease underwent positron emission tomography (PET) scanning with tracers specific for fibroblast activation protein (FAP; 68Ga-FAP inhibitor (FAPI)-04), 18F-fluorodeoxyglucose (FDG), MRI and histopathological assessment. In a longitudinal approach, 18F-FDG and 68Ga-FAPI-04 PET/CT data were evaluated before and after immunosuppressive treatment and correlated to clinical and MRI data. RESULTS: Using combination of 68Ga-FAPI-04 and 18F-FDG-PET, we demonstrate that non-invasive functional tracking of IgG4-related disease evolution from inflammatory towards a fibrotic outcome becomes feasible. 18F-FDG-PET positive lesions showed dense lymphoplasmacytic infiltration of IgG4+ cells in histology, while 68Ga-FAPI-04 PET positive lesions showed abundant activated fibroblasts expressing FAP according to results from RNA-sequencing of activated fibroblasts. The responsiveness of fibrotic lesions to anti-inflammatory treatment was far less pronounced than that of inflammatory lesions. CONCLUSION: FAP-specific PET/CT permits the discrimination between inflammatory and fibrotic activity in IgG4-related disease. This finding may profoundly change the management of certain forms of immune-mediated disease, such as IgG4-related disease, as subtypes dominated by fibrosis may require different approaches to control disease progression, for example, specific antifibrotic agents rather than broad spectrum anti-inflammatory treatments such as glucocorticoids.

Generation and characterization of hepatocellular carcinoma cell lines with enhanced cancer stem cell potential.

Hepatocellular carcinoma (HCC) is one of the most common causes for cancer-related death worldwide with rapidly increasing incidence and mortality rates. As for other types of cancers, also in HCC cancer stem cells (CSCs) are thought to be responsible for tumour initiation, progression and therapy failure. However, as rare subpopulations of tumour tissue, CSCs are difficult to isolate, thus making the development of suitable and reliable model systems necessary. In our study, we generated HepG2 subclones with enriched CSC potential by application of the spheroid formation method and subsequent single-cell cloning. Analyses in several 2D and 3D cell culture systems as well as a panel of functional assays both in vitro and in vivo revealed that the generated subclones displayed characteristic and sustained features of tumour initiating cells as well as highly aggressive properties related to tumour progression and metastasis. These characteristics could clearly be correlated with the expression of CSC markers that might have prognostic value in the clinical HCC setting. Therefore, we conclude that our CSC enriched HepG2 clones certainly represent suitable model systems to study the role of CSCs during HCC initiation, progression and drug resistance.

Influence of Cardiac MR Imaging on DNA Double-Strand Breaks in Human Blood Lymphocytes.

PURPOSE: To evaluate the ability of magnetic resonance (MR) imaging to induce deoxyribonucleic acid (DNA) damage in patients who underwent cardiac MR imaging in daily routine by using γ-H2AX immunofluorescence microscopy. MATERIALS AND METHODS: This study complies with the Declaration of Helsinki and was performed according to local ethics committee approval. Informed patient consent was obtained. Blood samples from 45 patients (13 women, 32 men; mean age, 50.3 years [age range, 20-89 years]) were obtained before and after contrast agent-enhanced cardiac MR imaging. MR imaging-induced double-strand breaks (DSBs) were quantified in isolated blood lymphocytes by using immunofluorescence microscopy after staining the phosphorylated histone variant γ-H2AX. Twenty-nine patients were examined with a myocarditis protocol (group A), 10 patients with a stress-testing protocol (group B), and six patients with flow measurements and angiography (group C). Paired t test was performed to compare excess foci before and after MR imaging. RESULTS: The mean baseline DSB level before MR imaging and 5 minutes after MR imaging was, respectively, 0.116 DSB per cell ± 0.019 (standard deviation) and 0.117 DSB per cell ± 0.019 (P = .71). There was also no significant difference in DSBs in these subgroups (group A: DSB per cell before and after MR imaging, respectively, 0.114 and 0.114, P = .91; group B: DSB per cell before and after MR imaging, respectively, 0.123 and 0.124, P = .78; group C: DSB per cell before and after MR imaging, respectively, 0.114 and 0.115, P = .36). CONCLUSION: By using γ-H2AX immunofluorescence microscopy, no DNA DSBs were detected after cardiac MR imaging.

Gain of a 500-fold sensitivity on an intravital MR contrast agent based on an endohedral gadolinium-cluster-fullerene-conjugate: a new chance in cancer diagnostics.

Among the applications of fullerene technology in health sciences the expanding field of magnetic resonance imaging (MRI) of molecular processes is most challenging. Here we present the synthesis and application of a Gd(x)Sc(3-x)N@C(80)-BioShuttle-conjugate referred to as Gd-cluster@-BioShuttle, which features high proton relaxation and, in comparison to the commonly used contrast agents, high signal enhancement at very low Gd concentrations. This modularly designed contrast agent represents a new tool for improved monitoring and evaluation of interventions at the gene transcription level. Also, a widespread monitoring to track individual cells is possible, as well as sensing of microenvironments. Furthermore, BioShuttle can also deliver constructs for transfection or active pharmaceutical ingredients, and scaffolding for incorporation with the host's body. Using the Gd-cluster@-BioShuttle as MRI contrast agent allows an improved evaluation of radio- or chemotherapy treated tissues.