Bradley H. Collins

The effect of soluble complement receptor type 1 on hyperacute rejection of porcine xenografts

The use of xenografts (Xgs) from distantly related species to relieve the increasing shortage of organs for clinical transplantation is prevented by the occurrence of hyperacute rejection (HAR). This process, in which C activation plays a central role, cannot be inhibited with currently available immunosuppressants. In two clinically relevant xenotransplantation models, this study evaluated the effect of C inhibition using recombinant soluble complement receptor type 1 (sCR1) on HAR. In an ex vivo model in which porcine cardiac Xgs were perfused with human blood, cardiac function ceased within 34 min when the perfusate blood was untreated (n = 3). When the perfusate blood was treated with sCR1 (300 micrograms/ml), cardiac Xg function was maintained for up to 4 hr (n = 3). Immunohistologic examination of these Xgs demonstrated deposition of C3b/iC3b and C3d in Xgs perfused with untreated human blood but only C3d deposition in those Xgs perfused with sCR1-treated human blood. These findings are consistent with the cofactor activity of sCR1 for factor I-mediated degradation of deposited C3b/iC3b to C3d. Treatment with sCR1 also prevented the histopathologic changes of HAR observed when untreated blood was used as the perfusate. In an in vivo pig-to-primate heterotopic cardiac xenotransplantation model, in which porcine Xgs transplanted into untreated cynomolgus monkey recipients underwent HAR in 1 hr or less (n = 3), a single intravenous bolus of sCR1 (15 mg/kg) administered to the recipient immediately before Xg reperfusion markedly inhibited total and alternative pathway serum C activity and prolonged Xg survival to between 48 and 90 hr (n = 5). These studies confirm the important role of C activation in HAR of porcine cardiac Xgs by primates and indicate that sCR1 may be a useful agent for xenotransplantation.

Immunoglobulin prevents complement-mediated hyperacute rejection in swine-to-primate xenotransplantation.

Immunoglobulins regulate the complement system by activating complement on foreign surfaces and diverting reactive complement proteins away from autologous cell surfaces. Based on this model, we explored the ability of Ig to balance complement activation versus control in a pig-to-primate cardiac xenotransplantation model in which the binding of xenoreactive antibodies of the recipient to graft blood vessels and the activation of complement cause hyperacute rejection. Human IgG added to human serum caused a dose-dependent decrease in deposition of iC3b, cytotoxicity, and heparan sulfate release when the serum was incubated with porcine endothelial cells. This decrease was not caused by alteration in antibody binding or consumption of complement but presumably reflected decreased formation of C3 convertase on the endothelial cells. Infusion of purified human IgG into nonhuman primates prevented hyperacute rejection of porcine hearts transplanted into the primates. As expected, the transplants contained deposits of recipient Ig and C1q but not other complement components. The inhibition of complement on endothelial cell surfaces and in the xenotransplantation model supports the idea that IgG regulates the classical complement pathway and supports therapeutic use of that agent in humoral-mediated disease.

Beneficial effect of plasmapheresis and intravenous immunoglobulin on renal allograft survival of patients with acute humoral rejection.

Background. Acute humoral rejection (AHR) has been associated with enhanced graft loss. Our study compared the renal allograft survival of patients with AHR treated with plasmapheresis (PP) and intravenous immunoglobulin (IVIG) with allograft survival in patients with acute cellular rejection (ACR). Methods. We retrospectively analyzed all kidney transplants performed at our institution between January 1999 and August 2001 (n=286). Recipients were classified into three groups according to biopsy reports: AHR, ACR, or no rejection. The ACR group was further divided into early and late rejection (<90 and >90 days posttransplant, respectively). Results. After a mean follow-up of 569±19 days, the incidence of AHR was 5.6% (n=16). Recipient presensitization, delayed graft function, early rejection, and higher creatinine at diagnosis were characteristic of AHR. Most AHR patients (14/16) were treated with PP and IVIG. One patient received only IVIG, whereas another received only PP. All AHR patients were given steroid pulses, but only four received antilymphocyte therapy because of concomitant severe ACR. The ACR group comprised 43 patients (15%). One patient with mild rejection received no therapy, 20 improved with steroids alone, and 22 required additional antilymphocyte therapy. One-year graft survival by Kaplan Meier analysis was 81% and 84% in the AHR and ACR groups, respectively (P =NS). Outcomes remained similar when AHR patients were compared with those with early ACR. Conclusions. We conclude that AHR, when diagnosed early and treated aggressively with PP and IVIG, carries a short-term prognosis that is similar to ACR.

The humoral immune response in humans following cross-perfusion of porcine organs

A major question in xenotransplantation is the nature of the humoral response that would occur following the transplantation of a xenogeneic organ into an immunosuppressed recipient as such a response could mediate delayed types of injury to the graft. To begin to address this issue we characterized the changes in the properties of xenoreactive antibodies occurring in patients exposed to porcine organs under conditions simulating transplantation. In two patients whose blood had been cross-perfused through porcine livers as a treatment for hepatic failure, the titer of xenoreactive IgM increased by four-fold and the titer of xenoreactive IgG increased by sixty-fold within ten days after perfusion procedures. The xenoreactive IgM and IgG antibodies were specific for Galα1–3Gal based on binding to porcine endothelial cells and bovine thyroglobulin, which express this determinant, and on the decrease in binding following treatment of porcine endothelial cells or bovine thyroglobulin with α-galactosidase. The sequential addition to endothelial cells of amounts of serum known to saturate antibody-binding sites obtained before and ten days after perfusion of porcine organs revealed no increase in binding of IgM above the level observed with serum obtained before perfusion, suggesting that new determinants were not identified. Moreover, the functional avidity of binding to porcine endothelial cells of IgM in serum obtained before and ten days after perfusion of porcine organs was unchanged. Even at later times, the presence of newly elicited antibodies against porcine aortic endothelial cell targets was not detected. Thus, exposure to porcine antigens in a vascularized organ results in increases in the levels of xenoreactive IgM and IgG antibodies—however, these antibodies exhibit properties similar to natural antibodies.

Characterization of porcine endothelial cell determinants recognized by human natural antibodies

Abstract: Organs transplanted from pigs to primates are subject to hyperacute rejection. This immunologic reaction is initiated by the recipients natural antibodies that bind to endothelial cell antigens of the organ, resulting in the activation of the complement system and rapid destruction of the graft. Various lines of evidence, particularly blocking studies, using purified carbohydrates have suggested that the endothelial cell determinant recognized by human natural antibodies is a terminal galactose in an α configuration (α‐Gal). Although these studies are compelling, they fall short of proof because xenoreactive natural antibodies, being polyreactive, might bind to structures other than those used for blocking. Moreover, in vivo evidence that anti‐α‐Gal antibodies participate in hyperacute rejection has not been reported. Here we report that an enzyme specific for α‐Gal, α‐galactosidase, removes α‐galactose residues from both intact porcine aortic endothelial cells and immobilized porcine aortic endothelial cell membrane extracts and that as a result of this, xenoreactive natural antibody binding is lowered by 70 to 80%. This decrease in binding of human IgM causes a corresponding decrease in complement activation. Similar results were obtained using the sera of other Old World primates. Digestion of immobilized porcine aortic endothelial cell membrane extracts with α‐galactosidase produced a similar reduction in human IgM binding to gp 115/135. These results indicate that a terminal α‐galactose is an important component of the gp 115/135 porcine endothelial cell antigen. We also report that perfusion of porcine organs I with primate serum removes anti‐α‐Gal IgM from the serum.

Mechanisms Of Injury In Porcine Livers Perfused With Blood Of Patients With Fulminant Hepatic Failure

Hyperacute rejection of renal and cardiac xenografts is initiated by the reaction of recipient natural antibodies and complement with endothelial cell antigens of the donor organ. The liver is thought to be less susceptible to this form of rejection; however, the mechanisms underlying its decreased susceptibility are not known. We investigated the organ injury occurring in porcine livers perfused with blood from 4 human subjects with fulminant hepatic failure. Nine porcine livers were perfused via an extracorporeal circuit in order to provide temporary metabolic support. Each porcine liver exhibited metabolic function, and the duration of xenoperfusion ranged from 2 to 5 hr. Histologic examination of the xenoperfused livers revealed focal hepatocellular necrosis, prominent infiltration of neutrophils, and, in 7 of 9 cases, periportal and centrilobular hemorrhage and thrombosis. Immunopathology demonstrated minimal or no human IgM and IgG along the small vessels and sinusoidal surfaces. Trace deposits of human IgM were observed along the luminal surfaces of large blood vessels in most cases. Trace deposits of C3 were noted in 2 of 9 livers; however, C4, iC3b, C5b, properdin, and the membrane attack complex were not detected. Human anti-porcine natural antibody titers decreased less than expected during the perfusions. Serum CH50, C3, and C4 levels were low before each procedure and decreased slightly with perfusion. One patient perfused 2 porcine livers and a human liver. The human liver

Effect of continuous complement inhibition using soluble complement receptor type 1 on survival of pig-to-primate cardiac xenografts

A single bolus of soluble complement (C) receptor type 1 (sCR1, TP-10) has been shown to delay hyperacute rejection (HAR) of porcine cardiac xenografts (Xgs) by primate recipients. In these recipients, C activity slowly returned and C deposition was noted in the Xgs at rejection. To evaluate the effect of sustained C inhibition using sCR1 on HAR, two additional cynomolgus monkeys received porcine cardiac Xgs and a continuous infusion of sCR1. In the first recipient, Xgs survival was 5 days (120+ hr), whereas in the second, Xg survival was 7 days (168+ hr). Serial biopsies of the Xgs were remarkable for an increasing cellular infiltrate composed predominantly of neutrophils and macrophages, and the development of edema, hemorrhage, and myocyte necrosis. These findings suggest that once C-mediated HAR has been inhibited, infiltration of the Xg by these cells may lead to accelerated acute rejection, which is an additional barrier to successful longer term Xg survival.

Microbial Biofilms in the Gut: Visualization by Electron Microscopy and by Acridine Orange Staining

The expression of colonization factors by gut bacteria, the growth rate of gut bacteria, and the rate of plasmid exchange by gut bacteria indicate that biofilms are a normal component of bacterial growth in the large bowel. Further, in vitro experiments demonstrate that growth of normal enteric bacteria in biofilms can be facilitated by secretory IgA (SIgA) and by mucins, 2 major components of the gut milieu. However, biofilms have not been previously observed in the normal gut. In this study, bacterial colonies characteristic of biofilms were observed by electron microscopy in normal rat, baboon, and human gut by electron microscopy. Confirming these results, acridine orange staining of flash-frozen tissues revealed biofilms in the mucus lining along normal gut epithelium. Immunofluorescenct microscopy supported this finding and demonstrated an association between IgA and the biofilms. These findings provide direct evidence that biofilms are present and may play an important role in the commensal relationship between enteric bacteria and their hosts. Hematoxylin and eosin staining of formalin-fixed tissues resulted in dissociation of the luminal contents from the epithelium, suggesting that the association between biofilms and the gut epithelium is sensitive to some conditions used to preserve tissue for histologic evaluation.

Peripheral vascular disease and renal transplant artery stenosis: a reappraisal of transplant renovascular disease

Background: Renal transplant artery stenosis (RTAS) continues to be a problematic, but potentially correctable, cause of post‐transplant hypertension and graft dysfunction. Older transplant recipients, prone to peripheral vascular disease (PVD), may have pseudoRTAS with PVD involving their iliac system.Methods: We retrospectively analyzed 819 patients who underwent kidney transplantation between 1993 and 1997 to determine the contribution of pseudoRTAS to renal transplant renovascular disease. Univariate analyses were performed for donor and recipient variables, including age, weight, gender, race, renal disease, cholesterol and creatinine values, human leukocyte antigen (HLA) matching, cytomegalovirus (CMV) infection, and immunosuppressive medications. Significant variables were then analyzed by a Cox proportional hazards model.Results: Ninety‐two patients (11.2%) underwent renal transplant arteriogram (Agram) or magnetic resonance angiography (MRA) for suspected RTAS. RTAS or pseudoRTAS, defined as one or more hemodynamically significant lesions in the transplant artery or iliac system, was evident in 44 patients (5.4%). Variables significantly associated with RTAS by univariate analysis were weight at the time of transplant (p=0.0258), male gender (p=0.034), discharge serum creatinine> 2 mg/dL (p=0.0041), and donor age (p=0.0062). Variables significantly associated with pseudoRTAS by univariate analysis were weight at the time of transplant (p=0.0285), recipient age (p=0.0049), insulin‐dependent diabetes mellitus (IDDM; p=0.0042), panel reactive antibody (PRA) at transplant (p=0.018), and body mass index (p=0.04). Weight at transplant and donor age remained significantly associated with an increased risk for RTAS in a multivariate stepwise Cox proportional hazards model. IDDM, transplant PRA, weight at transplant, and donor age were significantly associated with an increased risk for pseudoRTAS in a multivariate stepwise Cox proportional hazards model. Importantly, both RTAS and pseudoRTAS were associated with poorer graft survival (p<0.007 for each).Conclusions: Renal transplant renovascular disease encompasses pre‐existing PVD acting as pseudoRTAS, as well as classical RTAS. Efforts to identify and correct renal transplant renovascular disease of either nature are important, given its negative impact on graft survival.

Quality of life after treatment for pancreatitis.

OBJECTIVE The authors evaluated the morbidity, mortality, and quality of life after pancreatic debridement for necrosis and compared these values to those for quality of life after elective medical and surgical management for chronic pancreatitis. SUMMARY BACKGROUND DATA Quality of life after pancreatic debridement for necrosis has received little attention. Although quality of life after other pancreatic surgery has been evaluated and is though to be good, management of patients with pancreatic necrosis can be labor intensive and require extraordinary resources. Therefore, further evaluation of the quality of life achieved after treatment is appropriate. METHODS Forty patients (group 1) underwent operative debridement for necrosis between 1986 and 1994. Medical records of these patients were reviewed for morbidity, mortality, and in-hospital costs. Follow-up of quality of life was assessed by the Short Form-36 Health Survey. Patients in group 2 (n = 89) underwent medical management of chronic pancreatitis. Group 3 included 47 patients who underwent elective operations for ductal abnormalities. The Short Form-36 Health Surveys were administered to all three groups and compared statistically. RESULTS Mortality and morbidity from pancreatic debridement was 18% and 77%, respectively. Quality-of-life evaluations in groups 1 through 3 and age-matched controls were statistically similar. CONCLUSIONS Pancreatic debridement for necrosis requires intense application of resources and is associated with a high mortality and morbidity. Long-term follow-up shows good quality of life for patients who survive this morbid disease. This study supports the continued aggressive approach to the management of pancreatic necrosis, given that long-term outcome about quality of life is good.