We have examined processes leading to the spontaneous development of autoimmune inflammatory arthritis in transgenic mice containing CD4+ T cells targeted to a nominal Ag (hemagglutinin (HA)) and ...coexpressing HA driven by a MHC class II promoter. Despite being subjected to multiple tolerance mechanisms, autoreactive CD4+ T cells accumulate in the periphery of these mice and promote systemic proinflammatory cytokine production. The majority of mice spontaneously develop inflammatory arthritis, which is accompanied by an enhanced regional immune response in lymph nodes draining major joints. Arthritis development is accompanied by systemic B cell activation; however, neither B cells nor Ab is required for arthritis development, since disease develops in a B cell-deficient background. Moreover, arthritis also develops in a recombinase activating gene-deficient background, indicating that the disease process is driven by CD4+ T cells recognizing the neo-self HA Ag. These findings show that autoreactive CD4+ T cells recognizing a single self-Ag, expressed by systemically distributed APCs, can induce arthritis via a mechanism that is independent of their ability to provide help for autoantibody production.
Despite the impressive protection of B cell-deficient (muMT(-/-)) nonobese diabetic (NOD) mice from spontaneous diabetes, existence of mild pancreatic islet inflammation in these mice indicates that ...initial autoimmune targeting of beta cells has occurred. Furthermore, muMT(-/-) NOD mice are shown to harbor a latent repertoire of diabetogenic T cells, as evidenced by their susceptibility to cyclophosphamide-induced diabetes. The quiescence of this pool of islet-reactive T cells may be a consequence of impaired activation of T lymphocytes in B cell-deficient NOD mice. In this regard, in vitro anti-CD3-mediated stimulation demonstrates impaired activation of lymph node CD4 T cells in muMT(-/-) NOD mice as compared with that of wild-type counterparts, a deficiency that is correlated with an exaggerated CD4 T cell:APC ratio in lymph nodes of muMT(-/-) NOD mice. This feature points to an insufficient availability of APC costimulation on a per T cell basis, resulting in impaired CD4 T cell activation in lymph nodes of muMT(-/-) NOD mice. In accordance with these findings, an islet-reactive CD4 T cell clonotype undergoes suboptimal activation in pancreatic lymph nodes of muMT(-/-) NOD recipients. Overall, the present study indicates that B cells in the pancreatic lymph node microenvironment are critical in overcoming a checkpoint involving the provision of optimal costimulation to islet-reactive NOD CD4 T cells.
Systemic administration of soluble recombinant fusion protein of cytotoxic T lymphocyte antigen 4 (CTLA4Ig) induces blockade of the CD28/B7 costimulatory pathway and promotes survival of allogeneic ...and xenogeneic grafts. We tested the efficacy of local expression of CTLA4Ig gene in the myocardium, induced by transduction with a recombinant adenovirus encoding the CTLA4Ig gene, on the survival of rat cardiac allografts.
The donor hearts were perfused ex vivo with recombinant adenovirus encoding CTLA4Ig cDNA (AdCTLA4Ig) via intra-aorta coronary artery before transplantation. The distribution and duration of CTLA4Ig transgene expression in the myocardium was assessed by reverse transcriptase polymerase chain reaction (RT-PCR) or in situ RT-PCR after transplantation.
In situ RT-PCR demonstrated abundant expression of CTLA4Ig transgene in the endo-myocardium of AdCTLA4Ig-perfused cardiac grafts. Lewis and Brown Norway cardiac allografts transduced with AdCTLA4Ig survived indefinitely in nonimmunosuppressed Wistar Furth recipients. However, donor-strain skin grafts were rejected by long-term recipients of cardiac allografts, which also triggered the rejection of the primary heart grafts.
A single ex vivo intra-aortic infusion of recombinant adenovirus encoding the CTLA4Ig gene induced efficient transduction of the endo-myocardium and promoted the permanent survival of cardiac allografts in nonimmunosuppressed hosts. Despite the beneficial effect of local immunosuppression on cardiac allograft survival, the strategy failed to promote a state of donor-specific peripheral tolerance.
We previously demonstrated that T-regs inhibit proliferation of graft-reactive T cells in the draining lymph node (DLN), suggesting that this site may be important for regulation. TCR transgenic mice ...(TS1) specific for viral hemagglutinin (HA) provided antigen-specific T cells for adoptive transfer into syngeneic Balb/c hosts bearing HA+ skin grafts. T-regs were obtained from (TS1xHA28)F1 mice known to have an expanded population of HA-specific T-regs. To determine whether the lymph node is an independent site of suppression, we developed a model in which donor antigen that migrates from the allograft to the DLN drives T-cell activation after graft removal. T-regs that did not encounter the allograft itself remained able to inhibit graft antigen-specific T-cell proliferation in the DLN. Alloantigen-induced regulation can occur in the absence of the graft. This finding identifies the DLN as a potentially critical site of regulation in the early posttransplant period.
While great advances have been made in the success of islet transplantation to cure autoimmune diabetes, this protocol remains limited by our inability to induce donor‐specific tolerance within the ...recipient. The profound resistance of the NOD mouse to tolerance‐inducing regimens that are routinely successful in other strains further defines the imposing barriers that must be surmounted. Herein, we have assessed the utility of anti‐CD45RB therapy to induce tolerance to allografts in C57BL/6 and NOD‐strain mice. We find that, as with other therapies, NOD mice are also resistant to this manipulation, despite robust tolerance induction in the comparison strain. Analysis of cell surface markers revealed a number of changes within the B lymphocyte compartment following contact with antibody and alloantigen in the B6 strain. The absence of reciprocal changes within the NOD lymphocyte compartment suggests that B cells might contribute to the mechanism of action of this therapy and to the resistance to immunological tolerance noted in the NOD strain.
Objective: Uteroglobin is a protein with potent anti-inflammatory and immunomodulatory effects. We hypothesize that induction of uteroglobin expression in the artery wall by local adenoviral gene ...transfer will decrease neointimal hyperplasia in the rat carotid artery after balloon injury. Methods: Seven male Sprague-Dawley rats underwent balloon injury of the common carotid artery. After the injury, with flow occluded, the artery was instilled with 50 μL of the adenoviral vector encoding uteroglobin gene (Ad.UG) at a concentration of 1.35 × 1011 pfu/mL (n = 7) or 0.68 × 1011 pfu/mL (n = 7) (n = 7). Control animals were similarly treated: either an adenovirus encoding for β-galactosidase gene (Ad.LacZ) at 1 × 1011 pfu/mL (n = 7) or the phosphate-buffered saline (PBS) vehicle (n = 6) was used. The solution was allowed to dwell for 20 minutes. The rats were humanely killed after 14 days by perfusion fixation, and the carotid arteries were sectioned for analysis with computerized planimetry. The intima-media area ratios were calculated for each artery and compared with analysis of variance with Bonferroni/Dunn post hoc testing. One additional rat from the PBS, Ad.LacZ, and Ad.UG (1.35 × 1011 pfu/mL) groups was humanely killed 4 days after treatment for carotid artery protein extraction and Western blotting. Results: Uteroglobin protein production was confirmed in the Ad.UG-treated arteries with Western blotting. Morphometric analysis showed that the Ad.UG group at 1.35 × 1011 pfu/mL had a significantly lower intima-media area ratio than both the Ad.LacZ (P =.002) and PBS (P =.004) controls. The Ad.UG group at 0.68 × 1011 pfu/mL was also significantly different from the Ad.LacZ (P =.003) and PBS (P =.006) controls. There was no statistical difference between the two control groups or between the two Ad.UG groups. Conclusion: Adenoviral gene transfer of uteroglobin, delivered intraluminally after arterial injury causes the production of uteroglobin protein and has an inhibitory effect on neointimal accumulation in the rat model. (J Vasc Surg 2000;32:1111-7.)
Purpose. The aim of this study was to investigate the feasibility of gene transfer of uteroglobin, a potent anti-inflammatory and immunomodulatory agent, via adenoviral mediated gene transfer to the ...adventitia in the mouse carotid ligation injury model and also to investigate the efficacy of uteroglobin in reducing neointimal hyperplasia.
Methods. Forty-five C57bl/6NHSD mice were anesthetized and left common carotid artery ligation was performed. Adenoviral vector encoding the uteroglobin gene (Ad.UG; 15 μl of 1.35 × 1011 pfu/mL) was applied to the adventitia of the injured artery in 16 mice. In our control groups, 16 mice received adenoviral vector encoding the β-galactosidase reporter gene (Ad.lacZ; 15 μl of 1.0 × 1011 pfu/mL) and 13 mice received PBS only. Six mice from each group were sacrificed at 4 days for carotid artery protein extraction and Western blot analysis. The remainder were harvested at 30 days for histologic and morphometric analysis. The intima/media area ratios were calculated for each artery. The results were analyzed and compared using ANOVA and Bonferroni/Dunn post hoc testing.
Results. Two mice from the LacZ group and one from the PBS group died before the 30-day endpoint. Uteroglobin expression was demonstrated in the Ad.UG treated arteries by Western blot analysis. Morphometric analysis demonstrated a statistically significant reduction in the intima/media area ratio of Ad.UG treated carotids compared to controls. There was a reduction of intima/media ratio with Ad. UG treatment of 68% compared to Ad.lacZ treatment (P < 0.0001) and 62% compared to PBS treatment (P = 0.0006). There was no statistical difference between the control groups.
Conclusion. Adenoviral mediated gene transfer via the adventitia is an effective mode of gene delivery. Adventitial uteroglobin gene transfer using an adenoviral vector induces uteroglobin protein production and significantly reduces neointimal hyperplasia in themouse carotid ligation injury model.
While great advances have been made in the success of islet transplantation to cure autoimmune diabetes, this protocol remains limited by our inability to induce donor-specific tolerance within the ...recipient. The profound resistance of the NOD mouse to tolerance-inducing regimens that are routinely successful in other strains further defines the imposing barriers that must be surmounted. Herein, we have assessed the utility of anti-CD45RB therapy to induce tolerance to allografts in C57BL/6 and NOD-strain mice. We find that, as with other therapies, NOD mice are also resistant to this manipulation, despite robust tolerance induction in the comparison strain. Analysis of cell surface markers revealed a number of changes within the B lymphocyte compartment following contact with antibody and alloantigen in the B6 strain. The absence of reciprocal changes within the NOD lymphocyte compartment suggests that B cells might contribute to the mechanism of action of this therapy and to the resistance to immunological tolerance noted in the NOD strain.
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