Ex vivo delivery of Mirococept: A dose-finding study in pig kidney after showing a low dose is insufficient to reduce delayed graft function in human kidney.

The complement system plays a pivotal role in the pathogenesis of ischemia-reperfusion injury in solid organ transplantation. Mirococept is a potent membrane-localizing complement inhibitor that can be administered ex vivo to the donor kidney prior to transplantation. To evaluate the efficacy of Mirococept in reducing delayed graft function (DGF) in deceased donor renal transplantation, we undertook the efficacy of mirococept (APT070) for preventing ischaemia-reperfusion injury in the kidney allograft (EMPIRIKAL) trial (ISRCTN49958194). A dose range of 5-25 mg would be tested, starting with 10 mg in cohort 1. No significant difference between Mirococept at 10 mg and control was detected; hence the study was stopped to enable a further dose saturation study in a porcine kidney model. The optimal dose of Mirococept in pig kidney was 80 mg. This dose did not induce any additional histological damage compared to controls or after a subsequent 3 hours of normothermic machine perfusion. The amount of unbound Mirococept postperfusion was found to be within the systemic dose range considered safe in the Phase I trial. The ex vivo administration of Mirococept is a safe and feasible approach to treat DGF in deceased donor kidney transplantation. The porcine kidney study identified an optimal dose of 80 mg (equivalent to 120 mg in human kidney) that provides a basis for further clinical development.

Mucosal tolerance induced by an immunodominant peptide from rat alpha3(IV)NC1 in established experimental autoimmune glomerulonephritis.

Experimental autoimmune glomerulonephritis (EAG), an animal model of Goodpasture's disease, can be induced in Wistar Kyoto (WKY) rats by immunization with the noncollagenous domain of the alpha 3 chain of type IV collagen, alpha3(IV)NC1. Recent studies have identified an immunodominant peptide, pCol (24-38), from the N-terminus of rat alpha3(IV)NC1; this peptide contains the major B- and T-cell epitopes in EAG and can induce crescentic nephritis. In this study, we investigated the mechanisms of mucosal tolerance in EAG by examining the effects of the nasal administration of this peptide after the onset of disease. A dose-dependent effect was observed: a dose of 300 microg had no effect, a dose of 1000 microg resulted in a moderate reduction in EAG severity, and a dose of 3000 microg produced a marked reduction in EAG severity accompanied by diminished antigen-specific, T-cell proliferative responses. These results demonstrate that mucosal tolerance in EAG can be induced by nasal administration of an immunodominant peptide from the N-terminus of alpha3(IV)NC1 and should be of value in designing new therapeutic strategies for patients with Goodpasture's disease and other autoimmune disorders.

Regression of Atherosclerosis in ApoE-/- Mice Via Modulation of Monocyte Recruitment and Phenotype, Induced by Weekly Dosing of a Novel "Cytotopic" Anti-Thrombin Without Prolonged Anticoagulation.

Background Anticoagulants induce atherosclerosis regression in animal models but exploiting this clinically is limited by bleeding events. Here we test a novel thrombin inhibitor, PTL060, comprising hirulog covalently linked to a synthetic myristoyl electrostatic switch to tether to cell membranes. Methods and Results ApoE-/- mice were fed chow or high-fat diets, before transplantation of congenic aortic segments or injection of PTL060, parental hirulog, control saline, or labeled CD11b positive cells. Aortic transplants from transgenic mice expressing anticoagulants on endothelium did not develop atherosclerosis. A single intravenous injection of PTL060, but not hirulog inhibited atheroma development by >50% compared with controls when assessed 4 weeks later. Mice had prolonged bleeding times for only one seventh of the time that PTL060 was biologically active. Repeated weekly injections of PTL060 but not hirulog caused regression of atheroma. We dissected 2 contributory mechanisms. First, the majority of CCR2+ (C-C chemokine receptor type 2+) monocytes recruited into plaques expressed CCR7 (C-C chemokine receptor type 7), ABCA1 (ATP-binding cassette transporter - 1), and interleukin-10 in PTL060 mice, a phenotype seen in <20% of CCR2+ recruits in controls. Second, after several doses, there was a significant reduction in monocyte recruits, the majority of which were CCR2-negative with a similar regression-associated phenotype. Regression equivalent to that induced by intravenous PTL060 was induced by adoptive transfer of CD11b+ cells pre-coated with PTL060. Conclusions Covalent linkage of a myristoyl electrostatic switch onto hirulog in PTL060 uncouples the pharmacodynamic effects on hemostasis and atherosclerosis, such that plaque regression, mediated predominantly via effects on monocytes, is accompanied by only transient anticoagulation.

Homeostatic proliferation of lymphocytes results in augmented memory-like function and accelerated allograft rejection.

Homeostatic proliferation is a normal physiological process triggered by lymphopenia to maintain a constant level of T cells. It becomes the predominant source of new T cells in adulthood after thymus regression. T cells that have undergone homeostatic proliferation acquire the memory phenotype, cause autoimmune disease, and are resistant to tolerance induction protocols. Transplantation is a rare example in which lymphopenia is deliberately induced for its immunosuppressive effect. However, it is not known whether the homeostatic proliferation that follows will have the opposite effect and accelerate rejection. We show that T cells that have undergone homeostatic proliferation acquire a memory phenotype, spontaneously skews toward the Th1 phenotype, even in the absence of antigenic stimulus. Interestingly, in contrast, the percentage of Foxp3(+) regulatory T cells increased by 28-fold following homeostatic proliferation. Using a mouse life-sustaining kidney transplant model, we showed that T cells that have gone through homeostatic proliferation in lymphopenic hosts transformed chronic rejection to acute rejection of a single MHC class II-mismatched kidney allograft. T cells that have undergone homeostatic proliferation consistently cause reliable rejection even when bona fide memory T cells cannot. These functional changes are long-lasting and not restricted to the acute phase of homeostatic proliferation. Our findings have important implications for tolerance induction or graft-prolonging protocols involving leukocyte depletion such as irradiation bone marrow chimera, T cell-depleting Abs, and lymphopenia induced by infections such as CMV and HIV.

Ultra-localization of Foxp3+ T cells within renal allografts shows infiltration of tubules mimicking rejection.

Kidney transplant recipients are monitored for rejection by measurement of serum creatinine and graft biopsies. Biopsy samples are evaluated according to the Banff classification, which states that infiltration of tubules by mononuclear cells is an indicator of acute rejection. However, regulatory T cells play a crucial role in the overall immune response and are also present within transplanted tissue. We hypothesize that infiltration of mononuclear cells within kidney grafts is not always associated with rejection, especially if a high proportion of this infiltrate is regulatory T cells. Using a life-sustaining mouse kidney transplant model, we found that mononuclear cell tubular infiltration can occur in both rejecting and tolerant grafts. However, tolerant kidney grafts demonstrated a higher and sustained level of Foxp3+ regulatory cells. Importantly, a significant proportion of these cells were found within tubules. In cases in which graft function was normal, these cells were not harmful to the kidney and could be said to be mimicking, rather than causing, rejection.