Robert C. Harland

The role of antibodies in acute vascular rejection of pig-to-baboon cardiac transplants.

Long-term success in xenotransplantation is currently hampered by acute vascular rejection. The inciting cause of acute vascular rejection is not yet known; however, a variety of observations suggest that the humoral immune response of the recipient against the donor may be involved in the pathogenesis of this process. Using a pig-to-baboon heterotopic cardiac transplant model, we examined the role of antibodies in the development of acute vascular rejection. After transplantation into baboons, hearts from transgenic pigs expressing human decay-accelerating factor and CD59 underwent acute vascular rejection leading to graft failure within 5 d; the histology was characterized by endothelial injury and fibrin thrombi. Hearts from the transgenic pigs transplanted into baboons whose circulating antibodies were depleted using antiimmunoglobulin columns (Therasorb, Unterschleisshein, Germany) did not undergo acute vascular rejection in five of six cases. Biopsies from the xenotransplants in Ig-depleted baboons revealed little or no IgM or IgG, and no histologic evidence of acute vascular rejection in the five cases. Complement activity in the baboons was within the normal range during the period of xenograft survival. In one case, acute vascular rejection of a xenotransplant occurred in a baboon in which the level of antidonor antibody rose after Ig depletion was discontinued. This study provides evidence that antibodies play a significant role in the pathogenesis of acute vascular rejection, and suggests that acute vascular rejection might be prevented or treated by therapies aimed at the humoral immune response to porcine antigens.

Incidence and impact of de novo donor-specific alloantibody in primary renal allografts.

Background To date, limited information is available describing the incidence and impact of de novo donor-specific anti–human leukocyte antigen (HLA) antibodies (dnDSA) in the primary renal transplant patient. This report details the dnDSA incidence and actual 3-year post-dnDSA graft outcomes. Methods The study includes 189 consecutive nonsensitized, non-HLA-identical patients who received a primary kidney transplant between March 1999 and March 2006. Protocol testing for DSA via LABScreen single antigen beads (One Lambda) was done before transplantation and at 1, 3, 6, 9, and 12 months after transplantation then annually and when clinically indicated. Results Of 189 patients, 47 (25%) developed dnDSA within 10 years. The 5-year posttransplantation cumulative incidence was 20%, with the largest proportion of patients developing dnDSA in the first posttransplantation year (11%). Young patients (18–35 years old at transplantation), deceased-donor transplant recipients, pretransplantation HLA (non-DSA)–positive patients, and patients with a DQ mismatch were the most likely to develop dnDSA. From DSA appearance, 9% of patients lost their graft at 1 year. Actual 3-year death-censored post-dnDSA graft loss was 24%. Conclusion We conclude that 11% of the patients without detectable DSA at transplantation will have detectable DSA at 1 year, and over the next 4 years, the incidence of dnDSA will increase to 20%. After dnDSA development, 24% of the patients will fail within 3 years. Given these findings, future trials are warranted to determine if treatment of dnDSA-positive patients can prevent allograft failure.

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.

Treatment of renal allograft polyoma BK virus infection with leflunomide.

Background. Polyoma BK virus produces an aggressively destructive nephropathy in approximately 3% to 8% of renal allografts, is associated with graft loss within one year in 35% to 67% of those infected and there is no therapy of proven efficacy. Leflunomide is an immune suppressive drug with anti viral activity in vitro and in animals. Methods. We treated twenty-six patients with biopsy proven NK virus nephropathy (BKN) with either leflunomide alone (n=17) or leflunomide plus a course of cidofovir (n=9) and followed them for six to forty months. Leflunomide was dosed to a targeted blood level of active metabolite, A77 1726, of 50 &mgr;g/ml to 100 &mgr;g/ml (150 &mgr;M to 300 &mgr;M). Response to treatment was gauged by serial determinations of viral load in blood and urine (PCR), serum creatinine, and repeat allograft biopsy. Results. In the 22 patients consistently sustaining the targeted blood levels of active drug, blood and urine viral load levels uniformly decreased over time (P<.001). Mean serum creatinine levels stabilized over the first six months of treatment, and with 12 months or more of follow-up in 16 patients the mean serum creatinine has not changed significantly from base line. Four patients who did not consistently have blood levels of active drug (A77 1726) above 40 &mgr;g/ml did not clear the virus until these levels were attained or cidofovir was added. Conclusions. Leflunomide inhibits Polyoma virus replication in vitro and closely monitored leflunomide therapy with specifically targeted blood levels appears to be a safe and effective treatment for Polyoma BK nephropathy.

Silastic cuffed catheters for hemodialysis vascular access: thrombolytic and mechanical correction of malfunction.

Silastic cuffed catheters are assuming a greater role in providing long-term vascular access for hemodialysis patients. However, catheter thrombosis, fibrin sheath formation, and catheter malposition are recurrent problems that reduce extracorporeal flow rates and shorten catheter life. We reviewed 163 consecutive episodes of catheter malfunction that occurred in 121 catheters in 88 patients over a 3.5-year period. Intraluminal instillation of urokinase was successful in reestablishing an extracorporeal flow rate of > or = 300 mL/min in 74% of episodes. The 42 remaining episodes (26%) were radiologically evaluated. Two catheters required replacement for catheter kinking or insufficient catheter length. Two additional catheters were malpositioned; both were successfully repositioned with percutaneous techniques. A fibrin sheath was detected encasing the catheter in 38 instances. The fibrin sheath was successfully stripped from the distal portion of the catheter in 36 of the 38 instances. Using endoluminal thrombolytic therapy and percutaneous mechanical techniques, we have extended the mean survival for catheters intended for permanent vascular access to 12.7 months and have allowed 95% of the catheters inserted for temporary use to reach their use goal. Tunnel tract infection and catheter-mediated bacteremia were the primary reasons for catheter removal.

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.

The role of immunoglobulin-G subclasses and C1q in de novo HLA-DQ donor-specific antibody kidney transplantation outcomes.

Background Anti–HLA-DQ antibodies are the predominant HLA class II donor-specific antibodies (DSAs) after transplantation. Recently, de novo DQ DSA has been associated with worse allograft outcomes. The aim of this study was to determine the further complement-binding characteristics of the most harmful DQ DSA. Methods Single-antigen bead technology was used to screen 284 primary kidney transplant recipients for the presence of posttransplantation DQ DSA. Peak DSA sera of 34 recipients with only de novo DQ DSA and of 20 recipients with de novo DQ plus other DSAs were further analyzed by a modified single-antigen bead assay using immunoglobulin (Ig)-G subclass-specific reporter antibodies and a C1q-binding assay. Results Compared with recipients who did not have DSA, those with de novo persistent DQ-only DSA and with de novo DQ plus other DSAs had more acute rejection (AR) episodes (22%, P=0.005; and 36%, P=0.0009), increased risk of allograft loss (hazards ratio, 3.7, P=0.03; and hazards ratio, 11.4, P=0.001), and a lower 5-year allograft survival. De novo DQ-only recipients with AR had more IgG1/IgG3 combination and C1q-binding antibodies (51%, P=0.01; and 63%, P=0.001) than patients with no AR. Furthermore, the presence of C1q-binding de novo DQ DSA was associated with a 30% lower 5-year allograft survival (P=0.003). Conclusions The presence of de novo persistent, complement-binding DQ DSA negatively impacts kidney allograft outcomes. Therefore, early posttransplantation detection, monitoring, and removal of complement-binding DQ might be crucial for improving long-term kidney transplantation outcomes.

L-selectin and ICAM-1 mediate reperfusion injury and neutrophil adhesion in the warm ischemic mouse liver

Leukocytes recruited during ischemia-reperfusion to the liver are important mediators of injury. However, the mechanisms of leukocyte adhesion and the role of adhesion receptors in hepatic vasculature remain elusive. L-selectin may critically contribute to injury, priming adhesion for later action of intercellular adhesion molecule-1 (ICAM-1). Paired experiments were performed using mutant mice (L-selectin -/-, ICAM-1 -/-, and L-selectin/ICAM-1 -/-) and wild-type mice (C57BL/6) to investigate leukocyte adhesion in the ischemic liver. Leukocyte adhesion and infiltration were assessed histologically. Aspartate aminotransferase levels were significantly reduced (2- to 3-fold) in mutant vs. wild-type mice in most groups but most significantly after 90 min of partial hepatic ischemia. Leukocyte adhesion was significantly reduced in all mutant mice. Areas of microcirculatory failure, visualized by intravital microscopy, were prevalent in wild-type but virtually absent in L-selectin-deficient mice. After total hepatic ischemia for 75 or 90 min, survival was better in mutant L-selectin and L-selectin/ICAM-1 mice vs. wild-type mice and ICAM-1 mutants. In conclusion, L-selectin is critical in the pathogenesis of hepatic ischemia-reperfusion injury. Poor sinusoidal perfusion due to leukocyte adhesion and clot formation is a factor of injury and appears to involve L-selectin and ICAM-1 receptors.

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

Implications of Immunohistochemical Detection of C4d along Peritubular Capillaries in Late Acute Renal Allograft Rejection

Background. Immunohistochemical detection of the C4d complement product along peritubular capillaries (PC) may indicate humoral rejection of renal allografts. We examined the frequency of PC C4d expression in renal-allograft biopsies with acute rejection (AR) arising more than 6 months after transplantation and the impact of this finding. Methods. C4d was detected by immunoperoxidase in 2-micron paraffin sections of consecutive biopsies obtained over a 3-year period. The extent was classified as diffuse (≥50% PC C4d+), focal (<50% C4d+), and negative (C4d−). Clinical data were obtained by retrospective chart review. Fifty-five AR episodes with Banff 97 types 1A (n=13), 1B (n=26), 2A (n=11), 2B (n=3), and 3 (n=2) met inclusion criteria. Results. PC C4d expression was diffuse in 23 (42%), focal in 9 (16%), and negative in 23 (42%) biopsies. AR episodes with focal and diffuse C4d expression had higher proportionate elevation of serum creatinine at biopsy and 4 weeks after diagnosis (P≤0.05). Biopsies with diffuse PC C4d had interstitial hemorrhage (56.5%) and plasmacytic infiltrates (52%) more frequently than C4d− biopsies (22% and 16%), P=0.02, but had no other distinctive histologic features. Graft loss was greater in diffuse (65%) compared with focal C4d+ (33%) and C4d− (33%) groups 1 year after diagnosis, P=0.03. Other clinical and pathologic parameters did not differ significantly, including treatment received for AR. Conclusion. Evidence of acute cellular with occult humoral rejection is identified in more than 40% of late AR episodes. Late acute humoral rejection may be associated with interstitial hemorrhage and plasma cells and contributes significantly to graft loss.