Neal Smith

Heme oxygenase-1 affords protection against noncerebral forms of severe malaria

Infection by Plasmodium, the causative agent of malaria, is associated with hemolysis and therefore with release of hemoglobin from RBC. Under inflammatory conditions, cell-free hemoglobin can be oxidized, releasing its heme prosthetic groups and producing deleterious free heme. Here we demonstrate that survival of a Plasmodium-infected host relies strictly on its ability to prevent the cytotoxic effects of free heme via the expression of the heme-catabolyzing enzyme heme oxygenase-1 (HO-1; encoded by the Hmox1 gene). When infected with Plasmodium chabaudi chabaudi (Pcc), wild-type (Hmox1+/+) BALB/c mice resolved infection and restored homeostasis thereafter (0% lethality). In contrast, HO-1 deficient (Hmox1−/−) BALB/c mice developed a lethal form of hepatic failure (100% lethality), similar to the one occurring in Pcc-infected DBA/2 mice (75% lethality). Expression of HO-1 suppresses the pro-oxidant effects of free heme, preventing it from sensitizing hepatocytes to undergo TNF-mediated programmed cell death by apoptosis. This cytoprotective effect, which inhibits the development of hepatic failure in Pcc-infected mice without interfering with pathogen burden, is mimicked by pharmacological antioxidants such as N-acetylcysteine (NAC). When administered therapeutically, i.e., after Pcc infection, NAC suppressed the development of hepatic failure in Pcc-infected DBA/2 mice (0% lethality), without interfering with pathogen burden. In conclusion, we describe a mechanism of host defense against Plasmodium infection, based on tissue cytoprotection against free heme and limiting disease severity irrespectively of parasite burden.

Heme oxygenase-1 is essential for and promotes tolerance to transplanted organs

This investigation focused on obtaining a further understanding of the role of heme oxygenase‐1 (HO‐1) in tolerance induction. Hearts from C57BL/6 (H‐2b) mice survived long‐term when transplanted into BALB/c (H‐2d) recipients treated with the tolerance‐inducing regimen of anti‐CD40L antibody (MR‐1) plus donor‐specific transfusion (DST). Grafts did not, however, survive long‐term in (HO‐1−/−) recipients given the same treatment. Similarly, long‐term survival induced by DST was ablated when HO‐1 activity was blocked by zinc protoporphyrin IX (ZnPPIX). We further asked whether modulation of HO‐1 expression/activity could be used to promote the induction of graft tolerance. DST alone (day 0) failed to promote any prolongation of survival of DBA/2 (H‐2d) hearts transplanted into B6AF1 (H‐2b,k/d) recipients. However, long‐term survival and (dominant peripheral) tolerance were readily induced when DST was combined with induction of HO‐1 expression by cobalt protoporphyrin IX (CoPPIX). HO‐1 induction plus DST led to a significant up‐regulation of Foxp3, TGF‐β, IL‐10, and CTLA4, which suggests a prominent role for CD4+CD25+ regulatory T cells (Tregs). In fact, the tolerogenic effect of HO‐1 plus DST was dependent on CD4+CD25+ Tregs as suggested by adoptively transferring these cells into irradiated recipients under various regimens. Taken together, these findings show that expression of HO‐1 in a graft recipient can be essential for long‐term graft survival and for induction of tolerance and that modulation of HO‐1 expression/activity can be used therapeutically to synergize in the generation of graft tolerance.

Long-Term Survival of Hamster Hearts in Presensitized Rats

We transplanted hamster hearts into rats that had been sensitized to hamster cardiac grafts 5 days earlier as a model for discordant xenotransplantation. Sensitized rats had high serum levels of elicited anti-donor IgM and IgG that caused hyperacute rejection. Transient complement inhibition with cobra venom factor (CVF) plus daily and continuing cyclosporin A (CyA) prevented hyperacute rejection. However, grafts underwent delayed xenograft rejection (DXR). DXR involved IgG and associated Ab-dependent cell-mediated rejection, because depletion of IgG or Ab-dependent cell-mediated rejection-associated effector cells prolonged graft survival and the serum-mediated Ab-dependent cell-mediated cytotoxicity in vitro. Blood exchange in combination with CVF/CyA treatment dramatically decreased the level of preexisting Abs, but DXR still occurred in association with the return of Abs. Splenectomy and cyclophosphamide acted synergistically to delay Ab return, and when combined with blood exchange/CVF/CyA facilitated long-term survival of grafts. These grafts survived in the presence of anti-donor IgM, IgG, and complement that precipitated rejection of naive hearts, indicating that accommodation (survival in the presence of anti-graft Abs and complement) had occurred. We attribute the long-term survival to the removal of preexisting anti-donor Abs and therapy that attenuated the rate of Ab return. Under such conditions, the surviving hearts showed expression in endothelial cells and smooth muscle cells of protective genes and an intragraft Th2 immune response. Th2 responses and protective genes are associated with resistance to IgM- and IgG-mediated, complement-dependent and -independent forms of rejection.

Decay-accelerating factor prevents acute humoral rejection induced by low levels of anti-αGal natural antibodies

Background. Hyperacute and delayed vascular rejection due to natural antibodies (NAb) present major obstacles in pig-to-primate xenotransplantation. Although “supraphysiologic” expression of human complement regulatory proteins (CRPs) can prevent hyperacute rejection in discordant xenogenic recipients, their physiologic role in the homologous setting is undefined. We have evaluated the effect of the absence of decay-accelerating factor (DAF) on cardiac allograft rejection in the presence of different levels of antidonor antibodies (Ab). Methods. DAF1-deficient (DAF KO; B6129F2 H-2b) mice were used as heart graft donors to α1,3-galactosyltransferase deficient (GalT KO; B6, H-2b) recipients. Heterotopic heart grafting was performed with or without presensitization. Graft survival, histology, and anti-αGal Ab levels were monitored. Results. DAF knockout (KO) but not wild-type (WT) grafts showed hyperacute or acute humoral rejection in nonsensitized GalT KO mice with low levels of anti-αGal IgM NAb. However, humoral rejection of both DAF KO and DAF WT donor grafts occurred in presensitized GalT KO recipients. Conclusions. The expression of DAF prevents hyperacute rejection in mice with low titers of anti-αGal antibody. These studies demonstrate the physiologic role of DAF in preventing humoral rejection in the presence of low levels of NAb and have implications for transplantation of discordant vascularized xenografts.

Induction of xenograft accommodation by modulation of elicited antibody responses1 2.

Background. We have established that the timing of splenectomy influences the magnitude of the xenoreactive antibody (XAb) response and thus hamster heart survival in cyclosporine (CyA)-treated rats. This model has been used to test our hypothesis that modulation of XAb responses without perturbation of complement may influence the development of graft accommodation. Methods. Pretransplantation splenectomy (day −1/day 0) fully abrogated anti-graft IgM response, whereas a delayed procedure (day 1/day 2) caused significantly delayed (3–4 days) and decreased levels (two- to threefold) of XAb. Both interventions resulted in long-term graft survival. After surviving for 7 or more days, xenografts in CyA-treated rats with post-, but not pre-, transplantation splenectomy were also resistant to exogenous anti-graft XAb. Such grafts meet the criteria for accommodation. Accommodating hearts displayed progressive and increasing expression of protective genes, such as heme oxygense (HO)-1 and A20, in endothelial cells and smooth muscle cells. Results. Our results suggest that XAb responses may influence the kinetics of accommodation development possibly by promoting protective gene expression. This hypothesis was directly tested in vitro. Pretreatment of porcine aortic endothelial cells with sublytic amounts of baboon anti-pig serum for 24 hr induced HO-1 expression; this was associated with cell resistance to lytic amounts of such serum. Overexpression of HO-1 by adenoviral-mediated gene transfer in porcine aortic endothelial cells resulted in similar protective effects. Conclusions. Delayed and relatively low levels of XAb IgM promote expression of protective genes in the graft and thereby aid in the progress of accommodation. Expression of HO-1 protects xenoserum-mediated endothelial cell destruction.

Data-driven programmatic approach to analysis of basophil activation tests

Conventional data analysis of flow cytometry‐based basophil activation testing requires repetitive, labor‐intensive analysis that hampers efforts to standardize testing for clinical applications. Using an open‐source platform, we developed and implemented a programmatic approach to the analysis of the basophil activation test (BAT) by flow cytometry.