Continuous Acquisition of MHC:Peptide Complexes by Recipient Cells Contributes to the Generation of Anti-Graft CD8+ T Cell Immunity

Understanding the evolution of the direct and indirect pathways of allorecognition following tissue transplantation is essential in the design of tolerance‐promoting protocols. On the basis that donor bone marrow–derived antigen‐presenting cells are eliminated within days of transplantation, it has been argued that the indirect response represents the major threat to long‐term transplant survival, and is consequently the key target for regulation. However, the detection of MHC transfer between cells, and particularly the capture of MHC:peptide complexes by dendritic cells (DCs), led us to propose a third, semidirect, pathway of MHC allorecognition. Persistence of this pathway would lead to sustained activation of direct‐pathway T cells, arguably persisting for the life of the transplant. In this study, we focused on the contribution of acquired MHC‐class I on recipient DCs during the life span of a skin graft. We observed that MHC‐class I acquisition by recipient DCs occurs for at least 1 month following transplantation and may be the main source of alloantigen that drives CD8(+) cytotoxic T cell responses. In addition, acquired MHC‐class I:peptide complexes stimulate T cell responses in vivo, further emphasizing the need to regulate both pathways to induce indefinite survival of the graft

Paleoelevation estimates for the northern and central proto–Basin and Range from carbonate clumped isotope thermometry

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/99057/1/tect20016.pd

The Modular Socket System as Rural Solution in Indonesia

INTRODUCTION: The majority of the people in low-income countries, who need assistive technology do not have access to prosthetic devices [1]. Instead of these people having to make a long journey to one of the few prosthetic workshops, solutions like the Modular Socket System (MSS, Össur®) may be useful, because potentially they could be delivered and manufactured on site, at the location of the person [2]. This could make it suitable for application in a Community Based Rehabilitation (CBR) setting.The aim of this study was to evaluate the technical feasibility of the MSS for implementation in a CBR setting in terms of required tools, skills and required production time. METHODS: The study was performed at the Department of Prosthetics & Orthotics of the Jakarta I Polytechnic School of Health Science (JSPO). Four JSPO students received a three days training in manufacturing of the MSS. Lower limb amputees were recruited to participate in this study from the region of Jakarta (n = 5) and Bali (n = 10). A set of standardized instruments including the two minutes’ walking test (2MWT) and Prosthesis Evaluation Questionnaire (PEQ) were used to measure performance and satisfaction with the prosthesis. Production and maintenance logbooks were filled out by the involved prosthetists to evaluate the technical feasibility of the MSS. RESULTS AND DISCUSSION: Performance (2MWT) and satisfaction (PEQ) scores were comparable to that of similar studies with other lower leg prostheses [3,4]. Both measures did not decrease significantly over time (Figure 1). This suggest that the JSPO students were able to reach sufficient quality.It took the prosthetists 3.5 to 10.5 hours to fit an amputee with a MSS prosthesis. Mean socket production time was 2.0±0.6 hours and mean prosthesis assembly and fitting time was 4.1±2.6 hours. The only non-portable machine needed for the production of the prosthesis was a grinding machine (router). Smaller portable machines used were a cast cutter/jigsaw, Icecast® Compact and resin injection tool. If in the future the grinding machine will be replaced by a handheld tool, production of the MSS could be performed on site, making it suitable for use in a rural setting. Figure 1: The results of the 2MWT at the moment of fitting (t0), at 1-3 months post fitting (t1), and at the end evaluation at 4-6 months post fitting (t2). CONCLUSIONS: Patients who normally have to travel long distances to access prosthetic services were only required to make one visit to the health facility in order to receive a prosthesis. From a technical and quality perspective the method seems feasible, although, high costs remain an issue.ACKNOWLEDGEMENTSMaterials and training for the production of all prostheses were sponsored by Össur®. REFERENCES: 1.Borg J, et al. Assistive Technology for Children with Disabilities: Creating Opportunities for Education, Inclusion and Participation - a discussion paper. 20152.Normann E, et al., Prosthetics and orthotics international. 35(1):76-80, 20113.Boonstra AM, et al. Prosthetics and orthotics international. 17(2):78-82, 19934.Zidarov D, et al. Archives of Physical Medicine and Rehabilitation. 90(4):634-645, 200

Radiolabelling of Polyclonally Expanded Human Regulatory T Cells (Treg) with ⁸⁹Zr-oxine for Medium-Term In Vivo Cell Tracking

Regulatory T cells (Tregs) are a promising candidate cell therapy to treat autoimmune diseases and aid the longevity of transplanted solid organs. Despite increasing numbers of clinical trials using human Treg therapy, important questions pertaining to their in vivo fate, distribution, and function remain unanswered. Treg accumulation in relevant tissues was found to be crucial for Treg therapy efficacy, but existing blood-borne biomarkers are unlikely to accurately reflect the tissue state. Non-invasive Treg tracking by whole-body imaging is a promising alternative and can be achieved by direct radiolabelling of Tregs and following the radiolabelled cells with positron emission tomography (PET). Our goal was to evaluate the radiolabelling of polyclonal Tregs with ⁸⁹Zr to permit their in vivo tracking by PET/CT for longer than one week with current preclinical PET instrumentation. We used [⁸⁹Zr]Zr(oxinate)₄ as the cell-labelling agent and achieved successful radiolabelling efficiency of human Tregs spanning 0.1–11.1 Bq ⁸⁹Zr/Treg cell, which would be compatible with PET tracking beyond one week. We characterized the ⁸⁹Zr-Tregs, assessing their phenotypes, and found that they were not tolerating these intracellular ⁸⁹Zr amounts, as they failed to survive or expand in a ⁸⁹Zr-dose-dependent manner. Even at 0.1 Bq ⁸⁹Zr per Treg cell, while ⁸⁹Zr-Tregs remained functional as determined by a five-day-long effector T cell suppression assay, they failed to expand beyond day 3 in vitro. Moreover, PET imaging revealed signs of ⁸⁹Zr-Treg death after adoptive transfer in vivo. In summary, ⁸⁹Zr labelling of Tregs at intracellular radioisotope amounts compatible with cell tracking over several weeks did not achieve the desired outcomes, as ⁸⁹Zr-Tregs failed to expand and survive. Consequently, we conclude that indirect Treg labelling is likely to be the most effective alternative method to satisfy the requirements of this cell tracking scenario

Inhibition of thrombin on endothelium enhances recruitment of regulatory T cells during IRI and when combined with adoptive Treg transfer, significantly protects against acute tissue injury and prolongs allograft survival

Ischemia-reperfusion injury (IRI) amplifies T cell alloimmune responses after transplantation with thrombin playing a key pro-inflammatory role. To explore the influence of thrombin on regulatory T cell recruitment and efficacy we used a well-established model of IRI in the native murine kidney. Administration of the cytotopic thrombin inhibitor PTL060 inhibited IRI, and by skewing expression of chemokines (reducing CCL2 and CCL3 but increasing CCL17 and CCL22) increased the infiltration of M2 macrophages and Tregs. When PTL060 was combined with infusion of additional Tregs, these effects were further amplified. To test the benefits of thrombin inhibition in a transplant model, BALB/c hearts were transplanted into B6 mice with or without perfusion with PTL060 in combination with Tregs. Thrombin inhibition or Treg infusion alone led to small increments in allograft survival. However, the combined therapy led to modest graft prolongation by the same mechanisms as in renal IRI; graft survival was accompanied by increased numbers of Tregs and anti inflammatory macrophages, and reduced expression of pro-inflammatory cytokines. While the grafts succumbed to rejection associated with the emergence of alloantibody, these data suggest that thrombin inhibition within the transplant vasculature enhances the efficacy of Treg infusion, a therapy that is currently entering the clinic to promote transplant tolerance

Part Variation Modeling to Avoid Scrap Parts in Multi-stage Production Systems

Manufacturing systems for today's products are complex systems requiring a variety of different processes in order to be able to manufacture all necessary part features. This also applies to the production of rotating components, which have experienced increasing demand at the latest due to the growth in mobility. As in almost every manufacturing process, quality-reducing defects can occur due to deviations for example tool wear, which cannot always be avoided. Those, that have accumulated from previous process steps can cause the occurrence of superimposed defects. This leads to complex relationships between quality defects in the end product and the numerous parameters of the manufacturing processes. To remain competitive, production must be optimized in order to identify defects as early as possible, as well as their dependencies and variation patterns. The paper presents an approach to identify and model part variations within multi-stage production systems. Subsequently, based on a detected deviation, a downstream compensation strategy can be proposed at an early stage of the manufacturing process, which uses the capability of the overall system to fundamentally eliminate rejects

A two-tiered mechanism by which Cdc42 controls the localization and activation of an Arp2/3-activating motor complex in yeast

The establishment of cell polarity in budding yeast involves assembly of actin filaments at specified cortical domains. Elucidation of the underlying mechanism requires an understanding of the machinery that controls actin polymerization and how this machinery is in turn controlled by signaling proteins that respond to polarity cues. We showed previously that the yeast orthologue of the Wiskott-Aldrich Syndrome protein, Bee1/Las17p, and the type I myosins are key regulators of cortical actin polymerization. Here, we demonstrate further that these proteins together with Vrp1p form a multivalent Arp2/3-activating complex. During cell polarization, a bifurcated signaling pathway downstream of the Rho-type GTPase Cdc42p recruits and activates this complex, leading to local assembly of actin filaments. One branch, which requires formin homologues, mediates the recruitment of the Bee1p complex to the cortical site where the activated Cdc42p resides. The other is mediated by the p21-activated kinases, which activate the motor activity of myosin-I through phosphorylation. Together, these findings provide insights into the essential processes leading to polarization of the actin cytoskeleton

Nox2-deficient Tregs improve heart transplant outcomes via their increased graft recruitment and enhanced potency

Nox2 is a ROS-generating enzyme, deficiency of which increases suppression by Tregs in vitro and in an in vivo model of cardiac remodelling. Since Tregs have emerged as a candidate therapy in autoimmunity and transplantation, we hypothesised that Nox2 deficiency in Tregs in recipient mice may improve outcomes in a heart transplant model. A novel B6129 mouse model with Treg-targeted Nox2 deletion (Nox2ᶠˡ/ᶠˡFoxP3Cre⁺) was generated and transplanted with hearts from CB6F1 donors. As compared to littermate controls, Nox2ᶠˡ/ᶠˡFoxP3Cre⁺ mice had lower plasma levels of alloantibodies and troponin-I, reduced levels of IFN-γ in heart allograft homogenates and diminished cardiomyocyte necrosis and allograft fibrosis. Single cell analyses of allografts revealed higher absolute numbers of Tregs and lower CD8⁺ T cell infiltration in Nox2-deficient recipients compared to Nox2-replete mice. Mechanistically, in addition to a greater suppression of CD8⁺CD25⁻ T effector cell proliferation and IFN-γ production, Nox2-deficient Tregs expressed higher levels of CCR4 and CCR8, driving cell migration to allografts; this was associated with increased expression of miR214-3p. These data indicate that Nox2 deletion in Tregs enhances their suppressive ability and migration to heart allografts. Therefore, Nox2 inhibition in Tregs may be a useful approach to improve their therapeutic efficacy

B lymphocytes contribute to indirect pathway T cell sensitisation via acquisition of extracellular vesicles

B cells have been implicated in transplant rejection via antibody‐mediated mechanisms and more recently by presenting donor‐antigens to T cells. We have shown in patients with chronic antibody‐mediated rejection that B cells control the indirect T cell alloresponses. To understand more about the role of B cells as antigen presenting cells for CD4⁺ T cell with indirect allospecificity, B cells were depleted in C57BL/6 mice, using an anti‐CD20 antibody, prior to receiving MHC‐class I‐mismatched (Kᵈ) skin. The absence of B cells at the time of transplantation prolonged skin graft survival. To study the mechanisms behind this observation, T cells with indirect allospecificity were transferred in mice receiving a Kᵈ skin transplant. T cell proliferation was markedly inhibited in the absence of recipient B cells, suggesting that B cells contribute to indirect pathway sensitisation. Furthermore, we have shown that a possible way in which B cells present alloantigens is via acquisition of MHC‐peptide complexes. Finally, we demonstrate that the addition of B cell depletion to the transfer of Tregs with indirect alloresponse further prolonged skin graft survival. This study supports an important role for B cells in indirect T cell priming and further emphasises the advantage of combination therapies in prolonging transplant survival

Lower-limb amputees can reduce the energy cost of walking when assisted by an Active Pelvis Orthosis

Exoskeletons could compete with active prostheses as effective aids to reduce the increased metabolic demands faced by lower-limb amputees during locomotion. However, little evidence of their efficacy with amputees has been provided so far. In this paper, a portable hip exoskeleton has been tested with seven healthy subjects and two transfemoral amputees, with the final goal to verify whether a hip flexion-extension assistance could be effective in reducing the metabolic cost of walking. The metabolic power of the participants was estimated through indirect calorimetry during alternated repetitions of three treadmill-based walking conditions: without the exoskeleton (NoExo), with the exoskeleton in zero-torque mode (ExoTM) and with the exoskeleton providing hip flexion-extension assistance (ExoAM). The results showed that the exoskeleton reduced the net metabolic power of the two amputees in ExoAM with respect to NoExo, by 5.0% and 3.4%. With healthy subjects, a 5.5±3.1% average reduction in the metabolic power was observed during ExoAM compared to ExoTM (differences were not statistically significant), whereas ExoAM required 3.9±3.0% higher metabolic power than NoExo (differences were not statistically significant). These results provide initial evidence of the potential of exoskeletal technologies for assisting lower-limb amputees, thereby paving the way for further experimentations