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In &beta, thalassemia, ineffective erythropoiesis leads to anemia and systemic iron overload. The management of iron overload by chelation therapy is a standard of care. However, iron chelation does not improve the ineffective erythropoiesis. We recently showed that the oral ferroportin inhibitor VIT-2763 ameliorates anemia and erythropoiesis in the Hbbth3/+ mouse model of &beta, thalassemia. In this study, we investigated whether concurrent use of the iron chelator deferasirox (DFX) and the ferroportin inhibitor VIT-2763 causes any pharmacodynamic interactions in the Hbbth3/+ mouse model of &beta, thalassemia. Mice were treated with VIT-2763 or DFX alone or with the combination of both drugs once daily for three weeks. VIT-2763 alone or in combination with DFX improved anemia and erythropoiesis. VIT-2763 alone decreased serum iron and transferrin saturation (TSAT) but was not able to reduce the liver iron concentration. While DFX alone had no effect on TSAT and erythropoiesis, it significantly reduced the liver iron concentration alone and in the presence of VIT-2763. Our results clearly show that VIT-2763 does not interfere with the iron chelation efficacy of DFX. Furthermore, VIT-2763 retains its beneficial effects on improving ineffective erythropoiesis when combined with DFX in the Hbbth3/+ mouse model. In conclusion, co-administration of the oral ferroportin inhibitor VIT-2763 and the iron chelator DFX is feasible and might offer an opportunity to improve both ineffective erythropoiesis and iron overload in &beta, thalassemia.

β-Thalassemia is a genetic anemia caused by partial or complete loss of β-globin synthesis, leading to ineffective erythropoiesis and RBCs with a short life span. Currently, there is no efficacious oral medication modifying anemia for patients with β-thalassemia. The inappropriately low levels of the iron regulatory hormone hepcidin enable excessive iron absorption by ferroportin, the unique cellular iron exporter in mammals, leading to organ iron overload and associated morbidities. Correction of unbalanced iron absorption and recycling by induction of hepcidin synthesis or treatment with hepcidin mimetics ameliorates β-thalassemia. However, hepcidin modulation or replacement strategies currently in clinical development all require parenteral drug administration. We identified oral ferroportin inhibitors by screening a library of small molecular weight compounds for modulators of ferroportin internalization. Restricting iron availability by VIT-2763, the first clinical stage oral ferroportin inhibitor, ameliorated anemia and the dysregulated iron homeostasis in the Hbb(th3/+) mouse model of β-thalassemia intermedia. VIT-2763 not only improved erythropoiesis but also corrected the proportions of myeloid precursors in spleens of Hbb(th3/+) mice. VIT-2763 is currently being developed as an oral drug targeting ferroportin for the treatment of β-thalassemia.

Since the discovery of the first VLP derived from Hepatitis B virus in 19801, the field has expanded substantially. Besides successful use of VLPs as safe autologous virus-targeting vaccines, the powerful immunogenicity of VLPs has been also harnessed to generate immune response against heterologous and even self antigens2–4. Linking adjuvants to VLPs displaying heterologous antigen ensures simultaneous delivery of all vaccine components to the same antigen presenting cells. As a consequence, antigen presenting cells, such as dendritic cells (DCs) will process and present the antigen displayed on VLPs while receiving co-stimulatory signals by the VLP-incorporated adjuvant. Similarly, antigen-specific B cells recognizing the antigen linked to the VLP are simultaneously exposed to the adjuvant. Here we demonstrate in mice that physical association of antigen, carrier (VLPs) and adjuvant is more critical for B than T cell responses. As a model system, we used the E7 protein from human papilloma virus (HPV), which spontaneously forms oligomers with molecular weight ranging from 158 kDa to 10 MDa at an average size of 50 nm. E7 oligomers were either chemically linked or simply mixed with virus-like particles (VLPs) loaded with DNA rich in non-methylated CG motifs (CpGs), a ligand for toll-like receptor 9. E7-specific IgG responses were strongly enhanced if the antigen was linked to the VLPs. In contrast, both CD4+ and CD8+ T cell responses as well as T cell-mediated protection against tumor growth was comparable for linked and mixed antigen formulations. Therefore, our data show that B cell but not T cell responses require antigen-linkage to the carrier and adjuvant for optimal vaccination outcome.

Objective: The orally administered bacterial lysate OM-85 (Broncho-Vaxom®, Broncho-Munal®, Ommunal®, Paxoral®, Vaxoral®) is known to protect against recurrent respiratory tract infections. Despite mechanistic investigations performed during clinical and pre-clinical studies, little is known regarding the initial immune response to OM-85 following its passage through the stomach. To better understand the primary steps in the OM-85 gut-mediated immune-response cascade, we investigated its effects on two candidate cell types: intestinal epithelial cells (IECs) and Peyer’s patch (PP) leukocytes and confirmed its sustained immune effect in a reconstituted gastric buffer. Methods: In order to confirm OM-85’s continued activity following gastric transit, THP-1 cells were stimulated with OM-85 following its incubation in reconstituted gastric buffer (pH 1.7, 8 pM Pepsin). The ability of OM-85 to stimulate IECs was tested by incubating epithelial-cell lines (Caco-2 and HT-29) and freshly isolated mouse IEC aggregates with OM-85 or standard pattern-recognition receptor (PRR) ligands (Pam3CSK4, LPS, flagellin, or PGN). To test the ability of OM-85 to stimulate mucosal immune cells, PP cells isolated from mouse intestine were incubated with OM-85 or PRR ligands. Results: THP-1 cells released macrophage inflammatory protein-3 alpha (MIP-3α), both when OM-85 was pre-incubated in gastric buffer and when it was left untreated. In the presence of OM-85, functional PP cells freshly isolated from the intestine solely and dose-dependently released MIP-1α, a chemokine produced by myeloid cells which is involved in the recruitment and activation of various immune effector cells. Surprisingly, neither established epithelial-cell lines nor primary IECs of human or mouse origin released any of the tested cytokines in the presence of OM-85 or standard purified toll-like receptor (TLR)/nucleotide-binding oligomerisation domain (NOD)-like receptor ligands. Conclusion: These data suggested that primary mucosal PPs, but not IECs, are activated by ligands from the bacterial lysate OM-85. The secretion of MIP-1α from PPs might be a trigger signal inducing tonic stimulation of mucosal tissues to prepare host immune defense towards invading pathogens.

The efficiency of a vaccine largely depends on the appropriate targeting of the innate immune system, mainly through prolonged delivery of antigens and immunomodulatory substances to professional antigen-presenting cells in the lymphoid environment. Particulate antigens, such as virus-like particles (VLP) induce potent immune responses. However, little is known about the relative importance of direct drainage of free antigen to lymph nodes (LN) versus cellular transport and the impact of particle size on the process. Here, we show that nanoparticles traffic to the draining LN in a size-dependent manner. Whereas large particles (500-2000 nm) were mostly associated with dendritic cells (DC) from the injection site, small (20-200 nm) nanoparticles and VLP (30 nm) were also found in LN-resident DC and macrophages, suggesting free drainage of these particles to the LN. In vivo imaging studies in mice conditionally depleted of DC confirmed the capacity of small but not large particles to drain freely to the LN and demonstrated that DC are strictly required for transport of large particles from the injection site to the LN. These data provide evidence that particle size determines the mechanism of trafficking to the LN and show that only small nanoparticles can specifically target LN-resident cells.

Fibroblast contamination can make establishing primary melanoma cell cultures from native biopsies a major challenge, due to fibroblasts overgrowing the melanoma cells. Standard protocols therefore enrich for highly proliferative melanoma cells that grow well in vitro but may not represent the full range of in vivo tumor heterogeneity. Here we apply conditional methods that more effectively retrieve melanoma cells by differential trypsinization or by inducing fibroblast senescence through contact inhibition, serum starvation or deprivation of adhesion. Simple mixing experiments of melanoma and fibroblast cells demonstrated the efficacy of the new protocols in retrieving slow-growing melanoma cells. Applying our protocols to 20 cultures that had failed to grow by conventional methods, we could retrieve 12 (60%) validated melanoma cell cultures. Further application of the protocols in the live-cell biobank of 124 early passage cultures significantly improved recovery rates from 13% using standard protocols to 70% overall for the new workflow.

The cover shows a colour series of mouse footpads injected with small fluorescent nanoparticles. The image depicts the afferent lymphatic vessels connecting the injection site with the draining popliteal lymph node and the more distal sciatic lymph node. Lymphatic vessels (in green in the left row) run together with the veins (darker curves) and carry free draining antigen to the lymph nodes. This image was provided by the authors of Manolova (pp. 1404–1413) , in which it is demonstrated that particle size critically influences the mode of antigen transport to the draining lymph nodes; DC are strictly required for transport of large (500–2000 nm) particles, whereas smaller particles (20–200 nm), such as virus-like particles (30 nm), drain freely.