Direct recognition of hepatocyte-expressed MHC class I alloantigens is required for tolerance induction.

Adeno-associated viral vector-mediated (AAV-mediated) expression of allogeneic major histocompatibility complex class I (MHC class I) in recipient liver induces donor-specific tolerance in mouse skin transplant models in which a class I allele (H-2Kb or H-2Kd) is mismatched between donor and recipient. Tolerance can be induced in mice primed by prior rejection of a donor-strain skin graft, as well as in naive recipients. Allogeneic MHC class I may be recognized by recipient T cells as an intact molecule (direct recognition) or may be processed and presented as an allogeneic peptide in the context of self-MHC (indirect recognition). The relative contributions of direct and indirect allorecognition to tolerance induction in this setting are unknown. Using hepatocyte-specific AAV vectors encoding WT allogeneic MHC class I molecules, or class I molecules containing a point mutation (D227K) that impedes direct recognition of intact allogeneic MHC class I by CD8+ T cells without hampering the presentation of processed peptides derived from allogeneic MHC class I, we show here that tolerance induction depends upon recognition of intact MHC class I. Indirect recognition alone yielded a modest prolongation of subsequent skin graft survival, attributable to the generation of CD4+ Tregs, but it was not sufficient to induce tolerance.

Blockade of HMGB1 Attenuates Diabetic Nephropathy in Mice.

Activation of TLR2 or TLR4 by endogenous ligands such as high mobility group box 1 (HMGB1) may mediate inflammation causing diabetic kidney injury. We determined whether blockade of HMGB1 signaling by: (1) supra-physiological production of endogenous secretory Receptor for Advanced Glycation End-products (esRAGE), a receptor for HMGB1; (2) administration of HMGB1 A Box, a specific competitive antagonist, would inhibit development of streptozotocin induced diabetic nephropathy (DN). Wild-type diabetic mice developed albuminuria, glomerular injuries, interstitial fibrosis and renal inflammation. Using an adeno-associated virus vector, systemic over-expression of esRAGE afforded significant protection from all parameters. No protection was achieved by a control vector which expressed human serum albumin. Administration of A Box was similarly protective against development of DN. To determine the mechanism(s) of protection, we found that whilst deficiency of TLR2, TLR4 or RAGE afforded partial protection from development of DN, over-expression of esRAGE provided additional protection in TLR2-/-, modest protection against podocyte damage only in TLR4-/- and no protection in RAGE-/- diabetic mice, suggesting the protection provided by esRAGE was primarily through interruption of RAGE and TLR4 pathways. We conclude that strategies to block the interaction between HMGB1 and its receptors may be effective in preventing the development of DN.

Optimized method for ureteric reconstruction in a mouse kidney transplant model.

BACKGROUND: Murine kidney transplantation is an important model for studies of transplantation immunobiology. The most challenging aspect of the difficult surgical procedure is the ureteric anastomosis. METHODS: Two different approaches to ureteric reconstruction are compared here. Method 1, Patch: this involves anastomosis of the donor ureter together with a patch of donor bladder to recipient bladder. Method 2, Implant: this utilizes a 5-0 suture to pull the ureter through the bladder wall. The ureter's peripheral tissue is then fixed to the bladder wall at the implant site with 10-0 micro-sutures. RESULTS: In animals transplanted with the patch method, the initial success rate, defined as survival up to the third post-operative day, was 79% (n = 62), whereas the initial success rate for the implant method was 86.1% (n = 101; P = 0.28). The death rate from unknown and/or unspecified causes in the initial period was 16.1% (10/62) for the patch method, and 8.9% (9/101) for the implant method (P = 0.21). The average donor/recipient operation time with the implant method was 14.8 ± 2.2/61.4 ± 4.7 min (76 min per transplant), whereas operation time with the patch method was 28.3 ± 2.4/77.8 ± 5.5 min (106 min per transplant; P < 0.001). The ureteric implant method resulted in a lower rate of urinary leak compared with the patch method (1.1% versus 10.2%; P = 0.02). CONCLUSIONS: The ureteric implant method for mouse kidney transplantation is a reliable approach with at least as high a success rate as the bladder patch method and with a shorter operation time.

Liver transplant tolerance and its application to the clinic: can we exploit the high dose effect?

The tolerogenic properties of the liver have long been recognised, especially in regard to transplantation. Spontaneous acceptance of liver grafts occurs in a number of experimental models and also in a proportion of clinical transplant recipients. Liver graft acceptance results from donor antigen-specific tolerance, demonstrated by the extension of tolerance to other grafts of donor origin. A number of factors have been proposed to be involved in liver transplant tolerance induction, including the release of soluble major histocompatibility (MHC) molecules from the liver, its complement of immunosuppressive donor leucocytes, and the ability of hepatocytes to directly interact with and destroy antigen-specific T cells. The large tissue mass of the liver has also been suggested to act as a cytokine sink, with the potential to exhaust the immune response. In this review, we outline the growing body of evidence, from experimental models and clinical transplantation, which supports a role for large tissue mass and high antigen dose in the induction of tolerance. We also discuss a novel gene therapy approach to exploit this dose effect and induce antigen-specific tolerance robust enough to overcome a primed T cell memory response.

Gene therapy for tolerance: high-level expression of donor major histocompatibility complex in the liver overcomes naive and memory alloresponses to skin grafts.

BACKGROUND: The liver has long been recognized as having tolerogenic properties. We investigated whether recombinant adenoassociated virus (rAAV)-mediated expression of donor major histocompatibility complex in recipient livers could induce tolerance to donor-strain grafts. METHODS: Naive B10.BR (H-2) or B10.BR recipients primed with a H-2K-expressing (K) skin graft were injected with rAAV-expressing H-2K (rAAV-K) to induce K expression on hepatocytes 7 days before challenge with a K skin graft. K-specific responses were measured by interferon (IFN)-γ ELISpot and flow cytometric assessment of directly H-2K reactive cells. Fully allogeneic grafts from C57BL/6 (H-2) donors were transplanted onto longstanding B10.BR recipients of K skin to test for linked epitope suppression. RESULTS: rAAV-K-treated B10.BR mice accepted K skin grafts with increased median survival time (MST) more than 169 days compared to uninoculated (MST=18.5 days) and rAAV-K-treated controls (MST=19 days). rAAV-K-treated B10.BR animals primed with K skin grafts also accepted secondary K skin grafts in the long term (MST>100 days) compared to accelerated rejection in primed, uninoculated mice (MST=12 days). Treatments did not induce liver pathology, assessed by serum alanine aminotransferase levels and histology. IFN-γ ELISpot analysis of splenocytes from rAAV-K-treated mice indicated reduced responses to donor K antigen, but protection was not extended to fully allogeneic C57BL/6 skin or heart grafts, even in recipients that had accepted K skin grafts in the long term. CONCLUSIONS: High-level expression of donor major histocompatibility complex in recipient livers promotes tolerance to skin allografts, even in animals primed to produce a memory response. This provides proof of concept for an approach using liver-targeted gene delivery for tolerance induction to donor antigen.

Expression of common gamma chain signalling cytokines and their receptors distinguishes rejection from tolerance in a rat organ transplant model.

BACKGROUND: Signalling through the cytokine common γ chain (γc) is crucial for survival of activated T cells. In its absence, severe combined immunodeficiency ensues and transplanted tissues are not rejected. METHODS: To determine whether differences in the availability of γc signalling cytokines correlate with rejection or acceptance, we examined expression of all γc signalling components in organs transplanted between PVG donors and DA recipients. In this combination hearts or kidneys are rejected in <10 days while livers survive >100 days. Expression of the γc cytokines IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21 and their receptors γc, IL-2Rα, IL-2Rß/IL-15Rß, IL-4Rα, IL-7Rα, IL-9Rα, IL-15Rα and IL-21Rα was determined by real-time PCR pre-transplant and on days 3, 5 and 7 after transplantation. RESULTS: Most increased after transplantation, although there were significantly lower levels of IL-2, IL-2Rα, IL-4 and IL-15Rα in tolerant livers compared to rejecting hearts or kidneys. IL-9 was only expressed in normal kidneys and decreased during rejection. IL-15 was constitutively expressed and did not change after transplantation. IL-21 and IL-21R increased in all transplanted organs to a similar extent. IL-7Rα in liver was considerably increased compared with heart or kidney, consistent with its known inverse relationship to global levels of γc signalling. CONCLUSIONS: In transplanted livers, acceptance is associated with low levels of all γc cytokines or receptors except IL-21. This is consistent with "dilution" of γc cytokines from a finite clone size of alloreactive T cells in livers, which are ten times larger than kidneys or hearts.

Chemical target validation studies of aminopeptidase in malaria parasites using alpha-aminoalkylphosphonate and phosphonopeptide inhibitors.

During its intraerythrocytic phase, the most lethal human malarial parasite, Plasmodium falciparum, digests host cell hemoglobin as a source of some of the amino acids required for its own protein synthesis. A number of parasite endopeptidases (including plasmepsins and falcipains) process the globin into small peptides. These peptides appear to be further digested to free amino acids by aminopeptidases, enzymes that catalyze the sequential cleavage of N-terminal amino acids from peptides. Aminopeptidases are classified into different evolutionary families according to their sequence motifs and preferred substrates. The aminopeptidase inhibitor bestatin can disrupt parasite development, suggesting that this group of enzymes might be a chemotherapeutic target. Two bestatin-susceptible aminopeptidase activities, associated with gene products belonging to the M1 and M17 families, have been described in blood-stage P. falciparum parasites, but it is not known whether one or both are required for parasite development. To establish whether inhibition of the M17 aminopeptidase is sufficient to confer antimalarial activity, we evaluated 35 aminoalkylphosphonate and phosphonopeptide compounds designed to be specific inhibitors of M17 aminopeptidases. The compounds had a range of activities against cultured P. falciparum parasites with 50% inhibitory concentrations down to 14 muM. Some of the compounds were also potent inhibitors of parasite aminopeptidase activity, though it appeared that many were capable of inhibiting the M1 as well as the M17 enzyme. There was a strong correlation between the potencies of the compounds against whole parasites and against the enzyme, suggesting that M17 and/or M1 aminopeptidases may be valid antimalarial drug targets.

Characterization of the Plasmodium falciparum M17 leucyl aminopeptidase. A protease involved in amino acid regulation with potential for antimalarial drug development.

Amino acids generated from the catabolism of hemoglobin by intra-erythrocytic malaria parasites are not only essential for protein synthesis but also function in maintaining an osmotically stable environment, and creating a gradient by which amino acids that are rare or not present in hemoglobin are drawn into the parasite from host serum. We have proposed that a Plasmodium falciparum M17 leucyl aminopeptidase (PfLAP) generates and regulates the internal pool of free amino acids and therefore represents a target for novel antimalarial drugs. This enzyme has been expressed in insect cells as a functional 320-kDa homo-hexamer that is optimally active at neutral or alkaline pH, is dependent on metal ions for activity, and exhibits a substrate preference for N-terminally exposed hydrophobic amino acids, particularly leucine. PfLAP is produced by all stages in the intra-erythrocytic developmental cycle of malaria but was most highly expressed by trophozoites, a stage at which hemoglobin degradation and parasite protein synthesis are elevated. The enzyme was located by immunohistochemical methods and by transfecting malaria cells with a PfLAP-green fluorescent protein construct, to the cytosolic compartment of the cell at all developmental stages, including segregated merozoites. Amino acid dipeptide analogs, such as bestatin and its derivatives, are potent inhibitors of the protease and also block the growth of P. falciparum malaria parasites in culture. This study provides a biochemical basis for the antimalarial activity of aminopeptidase inhibitors. Availability of functionally active recombinant PfLAP, coupled with a simple enzymatic readout, will aid medicinal chemistry and/or high throughput approaches for the future design/discovery of new antimalarial drugs.