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.
Tertiary lymphoid organs are formed at sites of chronic inflammation and are thought to contribute to the immune response. Here, we aimed to characterize the structure and function of tertiary lymphoid organs in a model of murine kidney allotransplant to understand their role in alloimmunity.We transplanted 4 C57BL/6 mouse kidneys (isograft group) and 17 DBA/2 mouse kidneys into C57BL/6 mouse recipients. Three DBA/2-to-C57BL/6 transplant mice that rejected their grafts acutely (before 10 days posttransplant) were excluded from the study. The 14 surviving DAB2 grafts were retrieved at day 45 posttransplant and evaluated histologically. The presence of antibody-secreting cells and circulating levels of donor-specific antibodies were also evaluated.We found that tertiary lymphoid organs can be associated with a beneficial response in a kidney allotransplant model. Characterization of B-cell subsets within tertiary lymphoid organs in mouse kidney allografts revealed naive, plasma, and memory B cells, which were mostly grouped within or in close proximity of tertiary lymphoid organs. Staining for intracellular immunoglobulin G showed that many of the B cells within tertiary lymphoid organs were capable of producing antibodies. Although allospecific antibodies were found in the serum of recipient mice and were deposited in the transplanted kidneys, graft function was not affected in this model.B cells within tertiary lymphoid organs are functional and contribute to the humoral arm of the alloresponse. However, tertiary lymphoid organs are not necessarily associated with graft rejection, suggesting that protective mechanisms are at play.
Rejection of donor organs depends on the trafficking of donor passenger leukocytes to the secondary lymphoid organs of the recipient to elicit an immune response via the direct antigen presentation pathway. Therefore, the depletion of passenger leukocytes may be clinically applicable as a strategy to improve graft survival. Because major histocompatibility complex (MHC) class II(+) cells are most efficient at inducing immune responses, selective depletion of this population from donor grafts may dampen the alloimmune response and prolong graft survival. In a fully MHC mismatched mouse kidney allograft model, we describe the synthesis of an immunotoxin, consisting of the F(ab')2 fragment of a monoclonal antibody against the donor MHC class II molecule I-A(k) conjugated with the plant-derived ribosomal inactivating protein gelonin. This anti-I-A(k) gelonin immunotoxin depletes I-A(k) expressing cells specifically in vitro and in vivo. When given to recipients of kidney allografts, it resulted in indefinite graft survival with normal graft function, presence of Foxp3(+) cells within donor grafts, diminished donor-specific antibody formation, and delayed rejection of subsequent donor-type skin grafts. Strategies aimed at the donor arm of the immune system using agents such as immunotoxins may be a useful adjuvant to existing recipient-orientated immunosuppression.
Introduction: Short bowel syndrome is characterised by a severe reduction in the
amount of functional intestine available as an absorptive surface. Attempts to lengthen
the intestine by interposition of artificial tubular scaffolds juxtaposed between healthy
tissues have shown limited success. Transplantation is limited due to organ shortage.
The most promising solution may be implantation of tissue-engineered small intestine
using natural scaffold.
Materials and Results: Using a completely novel approach, up to 30cm lengths of
ileum with the attached vasculature were harvested from porcine donors. Separate
intestinal and vascular loops were identified and de-cellularised using individually
tailored detergent-enzymatic protocols. The resulting scaffold was compared to native
tissue in terms of retention of cellular and nuclear remnants, as well as structural and
functional proteins. Its biocompatibility was assessed by subcutaneous implantation of
1cm2 pieces into rat recipients. The remodeling fate of grafts was determined by time
related changes in the ratio of sub-populations of residual macrophages. Its
mechanical strength and ease of handling was evaluated by performing a left-sided
nephrectomy in an unrelated pig model, followed by end-to-end anastomosis of the
de-cellularised scaffolds’ mesenteric vasculature to the appropriate renal artery and
vein. In the last stage, porcine organoid units were isolated and their yield estimated
for future in vitro studies.
Conclusions: It is possible to simultaneously de-cellularise two different tissues of
varying cellular configuration and composition effectively and efficiently over a
relatively short period of time. The two key features of the de-cellularised scaffold are
that 1) the scaffold has in place the necessary architectural topography of small
intestine (including mucosal villi) and molecular cues for optimum re-cellularisation
and 2) the attached vascular tree provides an ideal conduit for re-cellularisation using
either vascular committed endothelial or progenitor cells. Ultimately, this scaffold can
be used for constructing long segments of bio-engineered intestine with the possibility
of immediate blood supply and re-vascularisation.
Background: Cardiac transplantation is an excellent treatment for end-stage heart disease. However, rejection of the donor graft, in particular, by chronic rejection leading to cardiac allograft vasculopathy, remains a major cause of graft loss. The lymphatic system plays a crucial role in the alloimmune response, facilitating trafficking of antigen-presenting cells to draining lymph nodes. The encounter of antigen-presenting cells with T lymphocytes in secondary lymphoid organs is essential for the initiation of alloimmunity. Donor lymphatic vessels are not anastomosed to that of the recipient during transplantation. The pathophysiology of lymphatic disruption is unknown, and whether this disruption enhances or hinders the alloimmune responses is unclear. Although histological analysis of lymphatic vessels in donor grafts can yield information on the structure of the lymphatics, the function following cardiac transplantation is poorly understood. Methods: Using single-photon emission computed tomography/computed tomography lymphoscintigraphy, we quantified the lymphatic flow index following heterotrophic cardiac transplantation in a murine model of chronic rejection. Results: Ten weeks following transplantation of a minor antigen (HY) sex-mismatched heart graft, the lymphatic flow index was significantly increased in comparison with sex-matched controls. Furthermore, the enhanced lymphatic flow index correlated with an increase in donor cells in the mediastinal draining lymph nodes; increased lymphatic vessel area; and graft infiltration of CD4 + , CD8 + T cells, and CD68 + macrophages. Conclusions: Chronic rejection results in increased lymphatic flow from the donor graft to draining lymph nodes, which may be a factor in promoting cellular trafficking, alloimmunity, and cardiac allograft vasculopathy.
The balance between antioxidants, such as ascorbate (ASC) and glutathione, and oxidative reactive oxygen species (ROS) is known to play a pivotal role in the response of plant cells to abiotic stress. Here cell cultures of Arabidopsis thaliana were investigated with regard to their response to elevated levels of cadmium. At concentrations <100 μM, Cd induces a rapid and concentration-dependent H2O2 accumulation. This response could be inhibited by diphenylene iodonium (DPI, 20 μM). Reverse transcription-PCR analysis of three RBOH (respiratory burst oxidase homologues) genes showed an increased transcription of RBOHF after 15 min. No change in ASC concentration was observed during the first 3 h after Cd addition. In contrast, glutathione levels completely diminished within 1 h. This drop could be attributed to an increase in phytochelatin 4. At the plasma membrane, Cd further induced a significant decrease in dehydroascorbate (DHA) uptake activity (up to 90% inhibition after 4 h). This decrease is not present when cells are treated with LaCl3 before exposure to CdCl2. LaCl3 is a typical inhibitor of Ca channels and prevents Cd uptake in these cells as well as the Cd-induced ROS production. Therefore, these results appear to indicate that Cd uptake is a prerequisite for the change in DHA transport activity. However, DPI did not prevent the drop in DHA uptake activity present in Cd-treated Arabidopsis cells, indicating that this response seems to be independent of the Cd-induced H2O2 production.
'%3e%3cpath fill='%23012169' d='M0 0v30h60V0z'/%3e%3cpath stroke='%23fff' stroke-width='6' d='m0 0 60 30m0-30L0 30'/%3e%3cpath stroke='%23C8102E' stroke-width='4' d='m0 0 60 30m0-30L0 30' clip-path='url(%23b)'/%3e%3cpath stroke='%23fff' stroke-width='10' d='M30 0v30M0 15h60'/%3e%3cpath stroke='%23C8102E' stroke-width='6' d='M30 0v30M0 15h60'/%3e%3c/g%3e%3c/svg%3e)
