The WNT16 locus is a major determinant of cortical bone thickness and nonvertebral fracture risk in humans. The disability, mortality and costs caused by osteoporosis-induced nonvertebral fractures are enormous. We demonstrate here that Wnt16-deficient mice develop spontaneous fractures as a result of low cortical thickness and high cortical porosity. In contrast, trabecular bone volume is not altered in these mice. Mechanistic studies revealed that WNT16 is osteoblast derived and inhibits human and mouse osteoclastogenesis both directly by acting on osteoclast progenitors and indirectly by increasing expression of osteoprotegerin (Opg) in osteoblasts. The signaling pathway activated by WNT16 in osteoclast progenitors is noncanonical, whereas the pathway activated in osteoblasts is both canonical and noncanonical. Conditional Wnt16 inactivation revealed that osteoblast-lineage cells are the principal source of WNT16, and its targeted deletion in osteoblasts increases fracture susceptibility. Thus, osteoblast-derived WNT16 is a previously unreported key regulator of osteoclastogenesis and fracture susceptibility. These findings open new avenues for the specific prevention or treatment of nonvertebral fractures, a substantial unmet medical need.

BackgroundCancer immunotherapy can be potentiated by conditioning regimens such as cyclophosphamide, which reduces the level of regulatory T cells (tregs). However, myeloid suppressive cells are still remaining. Accordingly to previous reports, gemcitabine improves immune status of cancer patients. In this study, the role of gemcitabine was further explored to map its immunological target cells and molecules in patients with pancreatic cancer.MethodsPatient blood was investigated by flow cytometry and cytokine arrays at different time points during gemcitabine treatment.ResultsThe patients had elevated myeloid-derived suppressor cells (MDSCs), and Tregs at diagnosis. Myeloid cells were in general decreased by gemcitabine. The granulocytic MDSCs were significantly reduced while monocytic MDSCs were not affected. In vitro, monocytes responding to IL-6 by STAT3 phosphorylation were prevented to respond in gemcitabine medium. However, gemcitabine could not prevent STAT3 phosphorylation in IL-6-treated tumor cell lines. TGFβ-1 was significantly reduced after only one treatment and continued to decrease. At the same time, the effector T cell:Treg ratio was increased and the effector T cells had full proliferative capacity during the gemcitabine cycle. However, after a resting period, the level of suppressor cells and TGFβ-1 had been restored showing the importance of continuous conditioning.ConclusionsGemcitabine regulates the immune system in patients with pancreatic cancer including MDSCs, Tregs and molecules such as TGFβ-1 but does not hamper the ability of effector lymphocytes to expand to stimuli. Hence, it may be of high interest to use gemcitabine as a conditioning strategy together with immunotherapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-016-1037-z) contains supplementary material, which is available to authorized users.

The flagellum protein flagellin of Listeria monocytogenes is encoded by the flaA gene. Immediately downstream of flaA, two genes, cheY and cheA, encoding products with homology to chemotaxis proteins of other bacteria, are located. In this study we constructed deletion mutants with mutations in flaA, cheY, and cheA to elucidate their role in the biology of infection with L. monocytogenes. The ⌬cheY, ⌬cheA, and double-mutant ⌬cheYA mutants, but not ⌬flaA mutant, were motile in liquid media. However, the ⌬cheA mutant had impaired swarming and the ⌬cheY and ⌬cheYA mutants were unable to swarm on soft agar plates, suggesting that cheY and cheA genes encode proteins involved in chemotaxis. The ⌬flaA, ⌬cheY, ⌬cheA, and ⌬cheYA mutants (grown at 24°C) showed reduced association with and invasion of Caco-2 cells compared to the wild-type strain. However, spleens from intragastrically infected BALB/c and C57BL/6 mice showed larger and similar numbers of the ⌬flaA and ⌬cheYA mutants, respectively, compared to the wild-type controls. Such a discrepancy could be explained by the fact that tumor necrosis factor receptor p55 deficient mice showed dramatically exacerbated susceptibility to the wild-type but unchanged or only slightly increased levels of the ⌬flaA or ⌬cheYA mutant. In summary, we show that listerial flaA, cheY, and cheA gene products facilitate the initial contact with epithelial cells and contribute to effective invasion but that flaA could also be involved in the triggering of immune responses.

Pancreatic cancer is a severe indication with short expected survival despite surgery and/or combination chemotherapeutics. Checkpoint blockade antibodies are approved for several cancer indications, but pancreatic cancer has remained refractory. However, there are clinical data suggesting that stimulation of the CD40 pathway may be of interest for these patients. Oncolytic viruses armed with immunostimulatory genes represent an interesting approach. Herein, we present LOAd703, a designed adenovirus armed with trimerized and that activates the CD40 and 4-1BB pathways, respectively. As many cells in the tumor stroma, including stellate cells and the infiltrating immune cells, express CD40 and some 4-1BB, we hypothesize that LOAd703 activates immunity and simultaneously modulates the biology of the tumor stroma. Tumor, stellate, endothelial, and immune cells were infected by LOAd703 and investigated by flow cytometry, proteomics, and functional analyses. LOAd703-infected pancreatic cell lines were killed by oncolysis, and the virus was more effective than standard-of-care gemcitabine. In xenograft models, LOAd703 efficiently reduced established tumors and could be combined with gemcitabine for additional effect. Infected stellate and tumor cells reduced factors that promote tumor growth (Spp-1, Gal-3, HGF, TGFβ and collagen type I), while chemokines were increased. Molecules involved in lymphocyte migration were upregulated on infected endothelial cells. Dendritic cells were robustly stimulated by LOAd703 to produce costimulators, cytokines and chemokines, and such DCs potently expanded both antigen-specific T cells and NK cells. LOAd703 is a potent immune activator that modulates the stroma to support antitumor responses. .

CD40 is an interesting target in cancer immunotherapy due to its ability to stimulate T-helper 1 immunity via maturation of dendritic cells and to drive M2 to M1 macrophage differentiation. Pancreatic cancer has a high M2 content that has shown responsive to anti-CD40 agonist therapy and CD40 may thus be a suitable target for immune activation in these patients. In this study, a novel oncolytic adenovirus armed with a trimerized membrane-bound extracellular CD40L (TMZ-CD40L) was evaluated as a treatment of pancreatic cancer. Further, the CD40L mechanisms of action were elucidated in cancer models. The results demonstrated that the virus transferring TMZ-CD40L had oncolytic capacity in pancreatic cancer cells and could control tumor progression. TMZ-CD40L was a potent stimulator of human myeloid cells and T-cell responses. Further, CD40L-mediated stimulation increased tumor-infiltrating T cells in vivo, which may be due to a direct activation of endothelial cells to upregulate receptors for lymphocyte attachment and transmigration. In conclusion, CD40L-mediated gene therapy is an interesting concept for the treatment of tumors with high levels of M2 macrophages, such as pancreatic cancer, and an oncolytic virus as carrier of CD40L may further boost tumor killing and immune activation.

Six 8-week-old Sprague-Dawley rats were studied for 9 days divided into three periods of 3 days each: before transferral to metabolism cages, during metabolic cage housing and after return to their home cages. Faeces were collected daily when the animals were housed in their home cages and every 6 h when the animals were housed in metabolic cages during which time urine was also collected every 6 h. The rate of weight gain was slightly reduced during the 3 days in metabolic cages and the animals produced significantly larger amounts of faeces when housed in metabolic cages than when housed in their home cages. The total faecal excretion of corticosterone (nanograms excreted per hour per kilogram body weight) and immunoglobulin A (IgA) (milligrams excreted per hour per kg body weight) quantified by enzyme-linked immunosorbent assays (ELISAs) exhibited a clear diurnal rhythm in the metabolic cage. Urinary excretions of corticosterone and IgA also followed a clear diurnal cycle. The mean daily amounts of corticosterone excreted were not significantly affected by cage change and by housing in metabolic cages. However, the excretion of faecal IgA was significantly reduced during the 3 days after the period in metabolic cages. Taken together the results indicate that metabolic cage housing is mildly stressful for young adult male rats.

ABSTRACT

Children exposed to systemic glucocorticoids often exhibit growth retardation and after the cessation of therapy catch-up growth occurs in many, but not all patients. The developmental regulation and underlying cellular mechanisms of catch-up growth are not fully understood. To clarify this issue, we established an in vitro model of catch-up growth. Here we present a protocol for the long-term culture (up to 160 days) of fetal (E20) as well as postnatal (P8) rat metatarsal bones which allowed us to characterize ex vivo the phenomenon of catch-up growth without any influence by systemic factors. The relevance of the model was confirmed by the demonstration that the growth of fetal and postnatal bones were stimulated by IGF1 (100 ng/ml) and inhibited by dexamethasone (Dexa; 1 mM). We found that the capacity to undergo catch-up growth was restricted to postnatal bones. Catch-up growth occurred after postnatal bones had been exposed to Dexa for 7 or 12 days but not after a more prolonged exposure (19 days). Incomplete catch-up growth resulted in compromised bone length when assessed at the end of the 4-month period of culture. While exposure to Dexa was associated with decreased chondrocyte proliferation and differentiation, catchup growth was only associated with increased cell proliferation. We conclude that the phenomenon of catch-up growth after Dexa treatment is intrinsic to the growth plate and primarily mediated by an upregulation of chondrocyte proliferation.