CD59a – A novel role in bone

The complement system has crucial functions in host defence. Novel data revealed a role for complement components in the pathology of osteoarthritis (OA). CD59a is a regulator of the terminal complement pathway in mice; the purpose of the study was to determine if CD59a-/- mice have an osteoarthritic bone phenotype. Osteoblast (OB) mineralisation, colony forming unit (CFU) and OCG assays were performed in vitro from bone marrow preparations of 8-20 week old mice. Decreased CFU differentiating towards osteoblasts and adipocytes (n=1 only), as well as an increased OCG, was revealed in male CD59a deficient (-/-) over wildtype (WT) mice. OCG in females were comparable. A human CD59 knockdown system utilising short hairpin (sh) ribonucleic acid (RNA) delivered by adenoviruses was established but did not differentiate into osteoclasts (OC). In vivo the bone phenotype of CD59a-/- mice was established for femora and vertebra L6 via X-ray, microcomputed tomography and histology. In male mice femoral length was increased in CD59a-/- versus WT mice at 8-10, 20 and 50 weeks. Cortical bone volume was increased whilst bone mineral density (BMD) was reduced in CD59a-/- versus WT mice at 8-10 and 20 weeks. Trabecular bone analysis of the distal femur (and spine) showed increased trabecular bone ratio, number, thickness, connectivity and total BMD in CD59a-/- over WT at 8-10 (and 20) weeks of age. In female mice there was no difference in femoral length and trabecular bone, but cortical BMD was raised at 50 weeks (CD59a-/- versus WT). Finally, histology revealed enhanced mineral apposition rate and OC surface as well as reduced osteoid surface in male CD59a-/- over WT mice at 8-10 weeks of age. Increased bone growth and turnover related to CD59a gene deletion were gender specific. These studies highlight CD59a as a potential target for OA treatment

Death receptor 3 (TNFRSF25) increases mineral apposition by osteoblasts and region specific new bone formation in the axial skeleton of male DBA/1 mice

Fraser L. Collins and this work were funded by an Arthritis Research UK PhD studentship (Grant Code: 18598) awarded to Anwen S. Williams, Eddie C. Y. Wang, and Michael D. Stone. Eddie C. Y. Wang was additionally funded by MRC Project Grant G0901119. Funding for open access was kindly provided by Cardiff University.Peer reviewedPublisher PD

CCL3 and MMP-9 are induced by TL1A during death receptor 3 (TNFRSF25)-dependent osteoclast function and systemic bone loss

FLC was funded by an Arthritis Research UK PhD studentship (Grant code: 18598) awarded to ASW, ECYW and MDS. JOW was funded by a British Heart Foundation PhD studentship (Reference: FS/11/26/ 28750). ACB's PhD studentship was jointly funded by the School of Medicine and Rheumatology Research Fund (Cardiff University) and LJ's PhD studentship was jointly funded by the School of Medicine and the President's Scholarship Fund (Cardiff University) awarded to ASW. ECYW was additionally funded by MRC Project Grant G0901119.Peer reviewedPublisher PD

Effect of modified vaccinia Ankara–5T4 and low-dose cyclophosphamide on antitumor immunity in metastatic colorectal cancer: A randomized clinical trial

Importance The success of immunotherapy with checkpoint inhibitors is not replicated in most cases of colorectal cancer; therefore, different strategies are urgently required. The oncofetal antigen 5T4 is expressed in more than 90% of cases of metastatic colorectal cancer (mCRC). Preliminary data using modified vaccinia Ankara–5T4 (MVA-5T4) in mCRC demonstrated that it safely induced serologic and T-cell responses. Objective To determine whether antitumor immunity in mCRC could be increased using MVA-5T4, metronomic low-dose cyclophosphamide, or a combination of both treatments. Design, Setting, and Participants In this randomized clinical trial, 55 patients with inoperable mCRC and prior stable disease after standard chemotherapy were enrolled at a single center and randomized to watch and wait (n = 9), cyclophosphamide treatment only (n = 9), MVA-5T4 only (n = 19), and a combination of MVA-5T4 and cyclophosphamide (n = 18). Patients were enrolled and treated from July 9, 2012, through February 8, 2016, and follow-up was completed on December 13, 2016. Data were analyzed based on intention to treat. Interventions Patients randomized to a cyclophosphamide group received 50 mg twice daily on treatment days 1 to 7 and 15 to 21. Patients randomized to a MVA-5T4 group received an intramuscular injection at a dose of 1 × 109 50% tissue culture infectious dose on treatment days 22, 36, 50, 64, 78, and 106. Main Outcomes and Measures The predefined primary end point was the magnitude of anti-5T4 immune responses (5T4-specific T-cell and antibody levels) generated at treatment week 7. Secondary end points included analysis of the kinetics of anti-5T4 responses, progression-free survival (PFS), and overall survival (OS). Results Fifty-two patients (38 men and 14 women; mean [SD] age, 64.2 [10.1] years) were included in the study analysis. The 5T4-specific antibody immune responses were significantly increased in the MVA-5T4 (83.41 [36.09] relative units [RU]; P = .02) and combination treatment (65.81 [16.68] RU; P = .002) groups compared with no treatment (20.09 [7.20] RU). Cyclophosphamide depleted regulatory T cells in 24 of 27 patients receiving MVA-5T4, independently prolonging PFS (5.0 vs 2.5 months; hazard ratio [HR], 0.48; 95% CI, 0.21-1.11; P = .09). MVA-5T4 doubled baseline anti-5T4 responses in 16 of 35 patients, resulting in significantly prolonged PFS (5.6 vs 2.4 months; HR, 0.21; 95% CI, 0.09-0.47; P < .001) and OS (20.0 vs 10.3 months; HR, 0.32; 95% CI, 0.14-0.74; P = .008). No grade 3 or 4 adverse events were observed. Conclusions and Relevance This initial randomized clinical immunotherapy study demonstrates a significant survival benefit in mCRC. Prior depletion of regulatory T cells by cyclophosphamide did not increase immune responses generated by MVA-5T4 vaccination; however, cyclophosphamide and MVA-5T4 each independently induced beneficial antitumor immune responses, resulting in prolonged survival without toxic effects. Larger clinical trials are planned to further validate these data

Low-dose cyclophosphamide induces anti-tumor T-cell responses which associate with survival in metastatic colorectal cancer

Purpose: Anti-cancer T-cell responses can control tumors, but immune-suppressive mechanisms in vivo prevent their function. The role of regulatory T-cells (Tregs) in metastatic colorectal cancer (mCRC) is unclear. We have previously shown depletion of Tregs enhances CRC-specific effector T-cell responses. Low dose cyclophosphamide (CPM) targets Tregs in animal models and some human studies, however the effect of CPM in mCRC is unknown. Experimental Design: Fifty-five mCRC patients were enrolled onto a phase I/II trial and randomized to receive two week-long courses of low-dose (50mg twice-a-day) CPM or not. The absolute number, phenotype and anti-tumor function of peripheral blood-derived lymphocyte subsets were monitored throughout treatment, along with 18-month follow-up. Results: Initially CPM reduced proliferation in all lymphocyte subsets, however, a rapid mobilization of effector T-cells overcame this decrease, leading to increased absolute T-cell numbers. In contrast, a reduction in proportional and absolute Treg, B-cell and NK-cell numbers occurred. The expansion and subsequent activation of effector T-cells was focused on tumor-specific T-cells, producing both granzyme B and IFN-gamma. CPM-treated patients demonstrating the most enhanced IFN-gamma+ tumor-specific T-cell responses exhibited a significant delay in tumor progression (HR=0.29, 95% CI 0.12-0.69, P=0.0047), compared to non-responders and no-treatment controls. Conclusions: CPM-induced Treg-depletion is mirrored by a striking boost to anti-tumor immunity. This study provides the first direct evidence of the benefit of naturally primed T-cells in mCRC patients. Our results also support the concept that non-mutated self-antigens can act as useful targets for immunotherapies

Primary breast tumours but not lung metastases induce protective anti-tumour immune responses after Treg-depletion

Although metastatic disease is responsible for the majority of cancer deaths, tests of novel immunotherapies in mouse tumour models often focus on primary tumours without determining whether these therapies also target metastatic disease. This study examined the impact of depleting Foxp3+ regulatory T cells (Treg), on lung metastases, using a mouse model of breast cancer. After Treg-depletion, generation of an immune response to the primary tumour was a critical determinant for limiting development of metastasis. Indeed, resection of the primary tumour abrogated any effect of Treg-depletion on metastases. In addition, whilst the immune response, generated by the primary tumour, prevented metastases development, it had little impact on controlling established disease. Collectively, the data indicate that metastatic cells in the lung are not controlled by immune responses induced by the primary tumour. These findings indicate that targeting Tregs alone will not suffice for treating lung metastases

Deletion of the membrane complement inhibitor CD59a drives age and gender-dependent alterations to bone phenotype in mice

Degenerative joint diseases such as osteoarthritis are characterised by aberrant region-specificboneformationand abnormal bone mineral content. A recent study suggested a role for the complement membrane attack com-plex in experimental models of osteoarthritis. Since CD59a is the principal regulator of the membrane attackcomplex in mice, we evaluated the impact of CD59a gene deletion upon maintenance of bone architecture.In vivobone morphology analysis revealed that male CD59a-deficient mice have increased femur length and cor-tical bone volume, albeit with reduced bone mineral density. However, this phenomenon was not observed infemale mice. Histomorphometric analysis of the trabecular bone showed increased rates of bone homeostasis,with both increased bone resorption and mineral apposition rate in CD59a-deficient male mice. When bonecells were studied in isolation,in vitroosteoclastogenesis was significantly increased in male CD59a-deficientmice, although osteoblast formation was not altered.Our data reveal, for thefirst time, that CD59a is a regulator of bone growth and homeostasis. CD59a ablation inmale mice results in longer and wider bones, but with less density, which is likely a major contributing factorfor their susceptibility to osteoarthritis. Thesefindings increase our understanding of the role of complementregulation in degenerative arthritis

Interleukin-6 limits influenza-induced inflammation and protects against fatal lung pathology

Balancing the generation of immune responses capable of controlling virus replication with those causing immunopathology is critical for the survival of the host and resolution of influenza-induced inflammation. Based on the capacity of interleukin-6 (IL-6) to govern both optimal T-cell responses and inflammatory resolution, we hypothesised that IL-6 plays an important role in maintaining this balance. Comparison of innate and adaptive immune responses in influenza-infected wild-type control and IL-6-deficient mice revealed striking differences in virus clearance, lung immunopathology and generation of heterosubtypic immunity. Mice lacking IL-6 displayed a profound defect in their ability to mount an anti-viral T-cell response. Failure to adequately control virus was further associated with an enhanced infiltration of inflammatory monocytes into the lung and an elevated production of the pro-inflammatory cytokines, IFN-α and TNF-α. These events were associated with severe lung damage, characterised by profound vascular leakage and death. Our data highlight an essential role for IL-6 in orchestrating anti-viral immunity through an ability to limit inflammation, promote protective adaptive immune responses and prevent fatal immunopathology

Cell Cycle-Dependent Microtubule-Based Dynamic Transport of Cytoplasmic Dynein in Mammalian Cells

BACKGROUND:Cytoplasmic dynein complex is a large multi-subunit microtubule (MT)-associated molecular motor involved in various cellular functions including organelle positioning, vesicle transport and cell division. However, regulatory mechanism of the cell-cycle dependent distribution of dynein has not fully been understood. METHODOLOGY/PRINCIPAL FINDINGS:Here we report live-cell imaging of cytoplasmic dynein in HeLa cells, by expressing multifunctional green fluorescent protein (mfGFP)-tagged 74-kDa intermediate chain (IC74). IC74-mfGFP was successfully incorporated into functional dynein complex. In interphase, dynein moved bi-directionally along with MTs, which might carry cargos such as transport vesicles. A substantial fraction of dynein moved toward cell periphery together with EB1, a member of MT plus end-tracking proteins (+TIPs), suggesting +TIPs-mediated transport of dynein. In late-interphase and prophase, dynein was localized at the centrosomes and the radial MT array. In prometaphase and metaphase, dynein was localized at spindle MTs where it frequently moved from spindle poles toward chromosomes or cell cortex. +TIPs may be involved in the transport of spindle dyneins. Possible kinetochore and cortical dyneins were also observed. CONCLUSIONS AND SIGNIFICANCE:These findings suggest that cytoplasmic dynein is transported to the site of action in preparation for the following cellular events, primarily by the MT-based transport. The MT-based transport may have greater advantage than simple diffusion of soluble dynein in rapid and efficient transport of the limited concentration of the protein