The type III intermediate filament vimentin regulates organelle distribution and modulates autophagy.

The cytoskeletal protein vimentin plays a key role in positioning of organelles within the cytosol and has been linked to the regulation of numerous cellular processes including autophagy, however, how vimentin regulates autophagy remains relatively unexplored. Here we report that inhibition of vimentin using the steroidal lactone Withaferin A (WFA) causes vimentin to aggregate, and this is associated with the relocalisation of organelles including autophagosomes and lysosomes from the cytosol to a juxtanuclear location. Vimentin inhibition causes autophagosomes to accumulate, and we demonstrate this results from modulation of mechanistic target of rapamycin (mTORC1) activity, and disruption of autophagosome-lysosome fusion. We suggest that vimentin plays a physiological role in autophagosome and lysosome positioning, thus identifying vimentin as a key factor in the regulation of mTORC1 and autophagy.

Lower resting and exercise-induced circulating angiogenic progenitors and angiogenic T cells in older men.

Aging is associated with a dysfunctional endothelial phenotype as well as reduced angiogenic capabilities. Exercise exerts beneficial effects on the cardiovascular system, possibly by increasing/maintaining the number and/or function of circulating angiogenic cells (CACs), which are known to decline with age. However, the relationship between cardiorespiratory fitness (CRF) and age-related changes in the frequency of CACs, as well as the exercise-induced responsiveness of CACs in older individuals, has not yet been determined. One-hundred seven healthy male volunteers, aged 18-75 yr, participated in study 1. CRF was estimated using a submaximal cycling ergometer test. Circulating endothelial progenitor cells (EPCs), angiogenic T cells (TANG), and their chemokine (C-X-C motif) receptor 4 (CXCR4) cell surface receptor expression were enumerated by flow cytometry using peripheral blood samples obtained under resting conditions before the exercise test. In study 2, 17 healthy men (8 young men, 18-25 yr; 9 older men, 60-75 yr) were recruited, and these participants undertook a 30-min cycling exercise bout at 70% maximal O2 consumption, with CACs enumerated before and immediately after exercise. Age was inversely associated with both CD34+ progenitor cells ( r2 = -0.140, P = 0.000) and TANG ( r2 = -0.176, P = 0.000) cells as well as CXCR4-expressing CACs (CD34+: r2 = -0.167, P = 0.000; EPCs: r2 = -0.098, P = 0.001; TANG: r2 = -0.053, P = 0.015). However, after correcting for age, CRF had no relationship with either CAC subset. In addition, older individuals displayed attenuated exercise-induced increases in CD34+ progenitor cells, TANG, CD4+, TANG, and CD8+CXCR4+ TANG cells. Older men display lower CAC levels, which may contribute to increased risk of cardiovascular disease, and older adults display an impaired exercise-induced responsiveness of these cells. NEW & NOTEWORTHY Older adults display lower circulating progenitor cell and angiogenic T cell counts compared with younger individuals independently of cardiometabolic risk factors and cardiorespiratory fitness. Older adults also display impaired exercise-induced mobilization of these vasculogenic cells.

Engineering Specificity and Function of Therapeutic Regulatory T Cells.

Adoptive therapy with polyclonal regulatory T cells (Tregs) has shown efficacy in suppressing detrimental immune responses in experimental models of autoimmunity and transplantation. The lack of specificity is a potential limitation of Treg therapy, as studies in mice have demonstrated that specificity can enhance the therapeutic potency of Treg. We will discuss that vectors encoding T cell receptors or chimeric antigen receptors provide an efficient gene-transfer platform to reliably produce Tregs of defined antigen specificity, thus overcoming the considerable difficulties of isolating low-frequency, antigen-specific cells that may be present in the natural Treg repertoire. The recent observations that Tregs can polarize into distinct lineages similar to the Th1, Th2, and Th17 subsets described for conventional T helper cells raise the possibility that Th1-, Th2-, and Th17-driven pathology may require matching Treg subsets for optimal therapeutic efficacy. In the future, genetic engineering may serve not only to enforce FoxP3 expression and a stable Treg phenotype but it may also enable the expression of particular transcription factors that drive differentiation into defined Treg subsets. Together, established and recently developed gene transfer and editing tools provide exciting opportunities to produce tailor-made antigen-specific Treg products with defined functional activities.

Remote ischaemic preconditioning in orthotopic liver transplantation (RIPCOLT trial): a pilot randomized controlled feasibility study.

BACKGROUND: Ischaemia Reperfusion (IR) injury is a major cause of morbidity, mortality and graft loss following Orthotopic Liver Transplantation (OLT). Utilising marginal grafts, which are more susceptible to IR injury, makes this a key research goal. Remote Ischaemic Preconditioning (RIPC) has been shown to ameliorate hepatic IR injury in experimental models. Whether RIPC can reduce IR injury in human liver transplant recipients is unknown. METHODS: Forty patients undergoing liver transplantation were randomized to RIPC or a sham. RIPC was induced through three 5 min cycles of alternate ischaemia and reperfusion of the left leg prior to surgery. Data on clinical outcomes was collected prospectively. Per-operative cytokine levels were measured. RESULTS: Fourty five of 51 patients approached (88%) were willing to enroll in the study. Five patients were excluded and 40 randomized, of which 20 underwent RIPC which was successfully completed in all patients. There were no complications following RIPC. Median day 3 AST levels were slightly higher in the RIPC group (221 IU vs 149 IU, p = 1.00). CONCLUSIONS: RIPC is acceptable and safe in liver transplant recipients. This study has not demonstrated evidence of a reduction in short-term measures of IR injury. Longer follow up will be required and consideration of an altered protocol.

A systematic review and meta-analysis of donor ischaemic preconditioning in liver transplantation.

Ischaemic preconditioning (IPC) is a strategy to reduce ischaemia-reperfusion (IR) injury. Its benefit in human liver transplantation is unclear. The aim of this study was to analyse the current evidence for donor IPC in liver transplantation. Systematic review and meta-analysis of studies involving IPC of liver transplant donors. Ovid Medline, Embase and Cochrane CENTRAL were searched up until January 2015. Data retrieved included the primary outcomes of 1-year mortality, incidence of primary graft non-function (PGNF) and retransplantation. Secondary outcomes included aspartate aminotransferase (AST) levels on day 3 post-op. Pooled odds ratios (ORs) were calculated for dichotomous data and mean weighted ratios for continuous data. Ten studies included 593 patients (286 IPC; 307 control). IPC was associated with a reduction in mortality at 1 year (6% vs. 11%) although this was not statistically significant (OR 0.54, 95% C.I. 0.28-1.04, P = 0.06). The IPC group had a significantly lower day 3 AST level (WMD -66.41iU, P = 0.04). This meta-analysis demonstrates that IPC reduces liver injury following transplantation and produces a large reduction in 1-year mortality which was not statistically significant. Confirmation of clinical benefit from IPC requires an adequately powered prospective RCT.

Protocol for a prospective randomized controlled trial of recipient remote ischaemic preconditioning in orthotopic liver transplantation (RIPCOLT trial).

ABSTRACT: Ischaemic reperfusion (IR) injury is a major cause of graft loss, morbidity and mortality following orthotopic liver transplantation (OLT). Demand for liver transplantation has resulted in increasing use of marginal grafts that are more prone to IR injury. Remote ischaemic preconditioning (RIPC) reduces IR injury in experimental models, but recipient RIPC has not been evaluated clinically. METHODS: A single-centre double-blind randomized controlled trial (RCT) is planned to test the hypothesis that recipient RIPC will reduce IR injury. RIPC will be performed following recipient anaesthetic induction but prior to skin incision. The protocol involves 3 cycles of 5 min of lower limb occlusion with a pneumatic tourniquet inflated to 200 mmHg alternating with 5 min of reperfusion. In the control group, the sham will involve the cuff being placed on the thigh but without being inflated. The primary endpoint is ability to recruit patients to the trial and safety of RIPC. The key secondary endpoint is a reduction in serum aspartate transferase levels on the third post-operative day. DISCUSSION: RIPC is a promising strategy to reduce IR injury in liver transplant recipients as there is a clear experimental basis, and the intervention is both inexpensive and easy to perform. This is the first trial to investigate RIPC in liver transplant recipients. TRIAL REGISTRATION: Clinicaltrials.gov NCT00796588.

Therapeutic potential of Tregs to treat rheumatoid arthritis.

There is accumulating evidence for regulatory T cell defects in rheumatoid arthritis and that some biologic interventions, in particular anti-TNF, can target this population. Despite the challenges in defining regulatory T cells in patients, there are a number of approaches currently being developed to utilise their potent immunosuppressive properties. Through genetic manipulation Tregs can be generated ex vivo or in vivo that target antigens present in the inflamed joint. Here we discuss these approaches, their refinement to restore tolerance in patients with rheumatoid arthritis, and strategies to prevent their conversion towards a Th17 phenotype.

CD3 limits the efficacy of TCR gene therapy in vivo.

The function of T-cell receptor (TCR) gene modified T cells is dependent on efficient surface expression of the introduced TCR α/ß heterodimer. We tested whether endogenous CD3 chains are rate-limiting for TCR expression and antigen-specific T-cell function. We show that co-transfer of CD3 and TCR genes into primary murine T cells enhanced TCR expression and antigen-specific T-cell function in vitro. Peptide titration experiments showed that T cells expressing introduced CD3 and TCR genes recognized lower concentration of antigen than T cells expressing TCR only. In vivo imaging revealed that TCR+CD3 gene modified T cells infiltrated tumors faster and in larger numbers, which resulted in more rapid tumor elimination compared with T cells modified by TCR only. After tumor clearance, TCR+CD3 engineered T cells persisted in larger numbers than TCR-only T cells and mounted a more effective memory response when rechallenged with antigen. The data demonstrate that provision of additional CD3 molecules is an effective strategy to enhance the avidity, anti-tumor activity and functional memory formation of TCR gene modified T cells in vivo.

Clinical applications of autoimmunity to citrullinated proteins in rheumatoid arthritis, from improving diagnostics to future therapies.

Rheumatoid arthritis (RA), although widely considered to be the most commonly occurring autoimmune disease, has only truly been substantiated as a distinct autoimmune disease very recently. The lack of understanding of the specific autoimmune system/s at work in rheumatoid patients resulted in an absence of robust diagnostic tools and had meant that the rational choice for use and design of therapy was based on broad-spectrum immunosuppression. The revelation that the autoimmune response specific for patients with RA is to particular protein antigens bearing the post-translational modification 'citrulline' has therefore revolutionized diagnostics and has helped explain why patients carrying particular MHC alleles are predisposed to the disease. The last two decades have seen the characterization of citrullinated antigens targeted by both antibodies and T cells in rheumatoid patients. In more recent years, we have also witnessed the success of biological therapies in the treatment of RA that specifically target T cells and B cells. Ongoing mapping of antibody targets is increasing the percentage of patients who can be definitively diagnosed with, and prognosed to develop, RA. These advances have led to a great number of patents for citrullinated peptides that have been and may be, in the coming years, used in diagnostic test kits. More recently, characterization of T cell targets (citrullinated peptides) has resulted in the patenting of peptides that could be used in antigen specific therapy. This review focuses on the characterization of the autoimmune response to citrullinated protein targets in RA and how the community is translating this knowledge to improve diagnostics, prognostics and therapy.

Natural IgM is required for suppression of inflammatory arthritis by apoptotic cells.

The clearance of dying cells is vital for re-establishing tolerance during inflammation and has potent immunoregulatory consequences. Because natural IgM plays a key role in the removal of apoptotic cells, we investigated whether the immune modulatory properties of apoptotic cells depended on its presence. Using an Ab-independent, Ag-induced model of inflammatory arthritis, we tested whether natural IgM is essential for the arthritis-suppressing properties of apoptotic cells. Whereas administration of apoptotic cells reduced joint inflammation and damage in normal mice accompanied by suppression of the Th17 response, no protection was afforded in secreted IgM-deficient (Sµ(-)) mice. The enhanced production of IL-10 by T cells from draining lymph nodes and splenic marginal zone B cells, driven by the infusion of apoptotic cells, was abrogated in the absence of natural IgM. Apoptotic cells were present shortly after administration in the splenic marginal zone, but their removal was substantially delayed in the absence of natural IgM. Incubation of apoptotic cells with natural IgM in vitro restored their arthritis-suppressing properties in Sµ(-) mice. Moreover, these IgM-coated apoptotic cells were cleared rapidly after injection from the spleens of Sµ(-) mice. Our results demonstrate that natural IgM is a critical factor in a chain of events triggered by the administration of apoptotic cells that promote IL-10-secreting B and T cells and restrain the development of inflammation.

Regulatory T-cell adoptive immunotherapy: potential for treatment of autoimmunity.

Tregs have shown considerable potential in treating preclinical models of autoimmunity. These studies have highlighted the importance of Treg antigen (Ag) specificity. Translation of these promising results to the clinic will require a robust method of generating large pure populations of Ag-specific Tregs. These include the recently described T-cell receptor gene transfer approach, which has proven to be a rapid and reliable method to generate large populations of Ag-specific Tregs. In this article, we will examine these various approaches and discuss their relative merits. Furthermore, we will discuss the obstacles that need to be surmounted to allow adoptive Treg therapy to progress to the clinic for the benefit of autoimmune patients.

Adoptive therapy with redirected primary regulatory T cells results in antigen-specific suppression of arthritis.

Regulatory T cells (Tregs) can suppress a wide range of immune cells, making them an ideal candidate for the treatment of autoimmunity. The potential clinical translation of targeted therapy with antigen-specific Tregs is hampered by the difficulties of isolating rare specificities from the natural polyclonal T cell repertoire. Moreover, the initiating antigen is often unknown in autoimmune disease. Here we tested the ability of antigen-specific Tregs generated by retroviral gene transfer to ameliorate arthritis through linked suppression and therefore without cognate recognition of the disease-initiating antigen. We explored two distinct strategies: T cell receptor (TCR) gene transfer into purified CD4+CD25+ T cells was used to redirect the specificity of naturally occurring Tregs; and co-transfer of FoxP3 and TCR genes served to convert conventional CD4(+) T cells into antigen-specific regulators. Following adoptive transfer into recipient mice, the gene-modified T cells engrafted efficiently and retained TCR and FoxP3 expression. Using an established arthritis model, we demonstrate antigen-driven accumulation of the gene modified T cells at the site of joint inflammation, which resulted in a local reduction in the number of inflammatory Th17 cells and a significant decrease in arthritic bone destruction. Together, we describe a robust strategy to rapidly generate antigen-specific regulatory T cells capable of highly targeted inhibition of tissue damage in the absence of systemic immune suppression. This opens the possibility to target Tregs to tissue-specific antigens for the treatment of autoimmune tissue damage without the knowledge of the disease-causing autoantigens recognized by pathogenic T cells.