The effect of soluble complement receptor type 1 on acute humoral xenograft rejection in hDAF-transgenic pig-to-primate life-supporting kidney xenografts

BACKGROUND: In pig-to-nonhuman primate solid organ xenotransplantation using organs from donors transgenic for human decay-accelerating factor (hDAF), the main type of rejection is antibody-mediated (acute humoral xenograft rejection, AHXR). This occurs despite the complement-regulatory function of the transgene, neutralization of natural antibodies to Galalpha1-3Gal (Gal) using soluble glycoconjugates, and chronic immunosuppression. As complement components play a major role in graft destruction after antibody binding, we evaluated the efficacy of chronic complement inhibition by soluble complement receptor type 1 (TP10). METHODS: Life-supporting hDAF-transgenic kidney transplantation was performed in cynomolgus monkeys, using cyclophosphamide induction, and maintenance immunosuppression with cyclosporin A, mycophenolate sodium, and tapering steroids. Rejection was treated with bolus steroid injections: if not successful animals were terminated. Three groups were studied: in group 1 (n=4) GAS914 (a soluble glycoconjugate comprising Gal on a poly-L-lysine backbone) was added before and after transplantation; group 2 (n=2) received GAS914 as in group 1 and in addition TP10 before and after transplantation; in group 3 (n=4) GAS914 was only given before transplantation and TP10 as in group 2. Monitoring included the regular assessment of anti-porcine antibodies, complement activity (soluble C5b-9), therapeutic drug monitoring, and graft histology. Results: Survival in group 1 was 6, 12, 31 and 37 days, respectively, and in all four cases graft histology showed AHXR. The two animals in groups 2 survived 3 and 15 days, respectively, and similarly showed AHXR in graft histology. In group 3 two animals showed AHXR (10 and 37 days survival, respectively), and two others did not show AHXR (20 and 32 days survival, respectively). The diagnosis AHXR included the deposition of complement activation products in the graft, which were present at lower intensity in animals treated with TP10. In all animals GAS914 effectively neutralized circulating anti-Gal antibody. Antibodies were detectable in the circulation of all animals using porcine erythrocytes in a hemolytic assay, although at lower levels than before transplantation. Soluble C5b-9 was not detectable in the circulation of animals receiving TP10, and circulating TP10 concentrations in these animals were in a presumed pharmacologically active range. CONCLUSIONS: The inclusion of TP10 in the immunosuppressive protocol does not clearly lead to improved xenograft survival. Despite effective neutralization of anti-Gal antibodies and effective inhibition of systemic complement activity, AHXR was apparent in four of six animals under chronic TP10 treatment, including deposits of complement activation products in the graft. Apparently, effective systemic complement inhibition by TP10 in combination with local complement regulation by the hDAF transgene product does not necessarily result in effective inhibition of complement activation at locations in the xenograft upon binding of anti-porcine antibodies to the grafted endothelium.     

Pathology of xenograft rejection: a commentary

Abstract: Trends in solid organ xenograft pathology are presented, with the focus on pig-to-nonhuman primate models. A simplified classification of rejection is followed, including hyperacute rejection (HAR), acute humoral xenograft rejection (AHXR), and acute cellular xenograft rejection (ACXR). The main components in HAR are natural xenoreactive antibodies in combination with complement activation. This is evident from the prevention of HAR in recipients in whom either antibodies or complement activation is depleted or inhibited. However, these strategies generally fail to prevent AHXR, which occurs later. AHXR is a multifactorial process in which natural and elicited antibodies may play roles, possibly in conjunction with complement, coagulation factors, and white blood cells. A main target appears to be the microvasculature which, in kidney grafts, is associated with a glomerular thrombotic microangiopathy. It is not clear to what extent species-specific physiologic disparities in complement and coagulation processes may play a role, separate from antibody-initiated processes. As rejection of solid organ xenografts is currently from AHXR, ACXR has not yet received close attention. In addition to intragraft rejection events, systemic complications following host–graft interactions have emerged, including (often fatal) consumptive coagulopathy and immune complex disease. It is anticipated that rejection processes will change when pigs with new genetic modifications become available. For instance, the precise role of natural antibodies to Galα1,3Gal will be able to be distinguished from other factors when pigs that lack the target antigen are available, and their organs can be evaluated in large animal xenotransplantation models.     

Characterization of baboon anti-porcine IgG antibodies during acute vascular rejection of porcine kidney xenograft

In the pig-to-baboon model, the removal of anti-porcine natural antibodies abrogates hyperacute vascular rejection (HAVR), but the xenograft then undergoes an acute vascular rejection (AVR) concomitantly to the appearance of newly formed anti-porcine antibodies. The use of anti-IgM monoclonal antibody (mAb) in baboons allowed to avoid HAVR of pig-to-baboon renal xenografts, but, at post-operative day 6, AVR occurred because of a rapid return of anti-porcine antibodies. The aim of this work was to characterize the anti-porcine antibodies during AVR. Sera from anti-IgM-treated animals were assessed prior to the graft and at the time of AVR by enzyme linked immunosorbent assay (ELISA) to determine anti-porcine antibodies concentration as well as the IgG subtypes. The same sera were tested on confluent cultures of porcine aortic endothelial cells (PAECs) to assess (i) the cytolytic complement-dependent activity and (ii) the E-selectin expression. The K affinity of anti-Gal IgG antibodies was measured by ELISA. Anti-porcine (Gal and non-Gal) IgG antibodies were tested on PAECs by flow cytometry to discriminate the presence of Gal epitopes from the recognition of other porcine epitopes. We found that both anti-porcine IgM and IgG antibodies presented a significantly increased cytolytic activity and E-selectin expression on PAECs during AVR. These characteristics are related to an important increase of the antibody (Ab) titer (especially anti-galactosyl) and a switch to anti-galactosyl IgG1 subclass production, whereas the K affinity remained unchanged. The deleterious effects of both IgM and IgG antibodies observed during AVR showed the crucial need for treatment controlling the cells producing anti-porcine antibodies.     

NK-cell-dependent acute xenograft rejection in the mouse heart-to-rat model

BACKGROUND: Acute humoral xenograft rejection is characterized by widespread intravascular thrombosis with a significant NK-cell and macrophage infiltrate. Although in vitro and ex vivo data have shown that NK cells are capable of killing xenogeneic tissue, the precise role they play in vivo is still not certain. Consequently, there are few tested strategies for dealing with NK-cell-mediated rejection, should this prove to be a problem. One reason for this has been the lack of a relevant rodent model in which rejection by these cells can be easily studied. METHODS: Prior to transplantation of mouse hearts, we depleted rat recipients of fibrinogen using a snake venom, ANCROD, from the Malayan pit viper. Graft survival was examined by manual palpation and the rejected hearts were examined by histology. Levels of circulating interferon gamma (IFN-gamma), used as a surrogate marker for NK-cell activation, were determined by an enzyme-linked immunosorbent assay. RESULTS: Depletion of fibrinogen to approximately 5% of normal allowed surgery without a significant increase in the technical failure rates and prolonged graft survival compared with that seen in unmanipulated rats. Rejected hearts showed no evidence of intravascular thrombosis but did show significant antibody and complement deposition. There was little T-cell infiltration and cyclosporin had no influence on survival. Instead, hearts were infiltrated with NK cells and macrophages and rejection was associated with significant IFN-gamma production. Depletion of NK cells with anti-asialo-GM-1 from ANCROD-treated recipients led to a further significant prolongation of graft survival. CONCLUSIONS: Inhibition of intravascular thrombosis by fibrinogen depletion, in the absence of any other manipulation, unmasks NK-cell-dependent acute xenograft rejection in the mouse-to-rat heart transplantation model. This relatively simple model is expected to be useful to investigate the mechanisms of NK-cell-mediated rejection and to provide insight into the types of graft manipulation that could modify this process.     

The graft content of hyaluronan is increased during xenograft rejection

BACKGROUND: Hyaluronan, a macromolecule with strong water binding capacity, is associated with interstitial oedema during rejection of allogeneic transplants. However, the involvement of hyaluronan during xenograft rejection has previously not been investigated. The aims of this study were to characterize hyaluronan content and distribution during rejection of concordant mouse-to-rat cardiac xenografts, and to explore the effects of hyaluronidase (HAse) on xenograft survival. METHODS: Graft recipients were treated with 15-deoxyspergualin (DSG) or both HAse and DSG. Grafts were removed on day 5 from some of the animals to analyse hyaluronan and water content, while other animals were used to investigate graft survival. The hyaluronan content was measured by a radiometric assay and the distribution was analysed by histochemical staining. RESULTS: In xenografts undergoing rejection (the DSG group) there was a strong increase of the hyaluronan [555 +/- 93 microg/g dry weight (dw)] and water (82.7 +/- 0.4%) contents compared with normal mouse heart tissue (166 +/- 10 microg/g dw; P < 0.01 and 78.6 +/- 0.5%; P < 0.001, respectively). The combined use of HAse and DSG reduced the accumulation of hyaluronan (284 +/- 43 microg/g dw; P < 0.05 vs. DSG) but did not affect the average water content. The average graft survival time did not differ between the groups; however, three grafts in the HAse + DSG-treatment group survived much longer than the longest-surviving grafts in the DSG group. CONCLUSIONS: These data suggest that the graft content of hyaluronan considerably increases during xenograft rejection. HAse effectively reduces this accumulation, but does not affect the average water content.     

Acute renal allograft rejection is associated with increased levels of vascular endothelial growth factor in the urine

AIM: The purpose of this study was to assess whether measurement of urinary vascular endothelial growth factor (VEGF) could be adopted as a new non-invasive diagnostic tool for acute rejection following renal transplantation. METHODS: Urinary concentration of VEGF was determined by an enzyme-linked immunosorbent assay technique in 215 renal allograft recipients and 80 healthy controls. RESULTS: Subjects with acute rejection (n=67) excreted urinary VEGF at a significantly higher level (28.57+/-6.21, 95% CI: 16.18-40.97 pg/mumol creatinine) than those without acute rejection. This included subjects with stable renal function and no abnormal histological findings (n=119), acute tubular necrosis (n=15), chronic allograft nephropathy (n=14) and healthy controls (n=80). Using a urinary VEGF/creatinine ratio of 3.64 pg/micromol as the cut-off point, the sensitivity and specificity for diagnosing acute rejection were 85.1 and 74.8%, respectively (P<0.001). Patients with steroid-resistant acute rejection had significantly greater urinary VEGF concentration than patients with steroid-sensitive acute rejection (42.09+/-10.00 vs 9.74+/-2.63 pg/micromol creatinine, P<0.001). Patients with graft loss after acute rejection had significantly greater urinary VEGF concentration than patients with reversible acute rejection (106.66+/-38.60 vs 19.46+/-4.13 pg/micromol creatinine, P=0.001). Using a urinary VEGF/creatinine ratio of 22.48 pg/micromol as the cut-off point, the sensitivity and specificity of the prediction to graft loss after acute rejection were 85.7% and 78.3%, respectively (P=0.001). CONCLUSION: This study demonstrates that the monitoring of urinary VEGF may be a useful non-invasive approach for the detection of acute rejection. Additionally, urinary VEGF levels were shown to predict the response to anti-rejection therapy and to predict a poor outcome after acute rejection.     

A case of acute vascular rejection after overseas deceased kidney transplantation

Abstract:  A 54-yr-old Japanese male received overseas deceased kidney transplantation in January 2006. His allograft functioned immediately and he received immunosuppression with cyclosporine A (CyA), mycophenolate mofetil (MMF), and prednisone (PR). On day 24 after transplantation, he came back to Japan. His serum creatinine level (s-Cr) was 1.39 mg/dL at two months after transplantation when he was admitted into Toda Central General Hospital on March 2006, for follow-up his renal allograft. He had taken only two immunosuppressive drugs, MMF and PR, and had not taken CyA at that time. His serum creatinine gradually rose after hospitalization. Allograft biopsy performed on April 6, 2006, showed acute vascular rejection (Banff 97 acute/active cellular rejection Grade III), together with suspicious for acute humoral rejection (Banff 97 antibody-mediated rejection Grade II). After treatment of two courses of steroid pulses and five d of gusperimus, acute vascular rejection and acute humoral rejection were relieved, which had been proven by the third allograft biopsy. In conclusion, this was a case of acute vascular rejection after overseas deceased kidney transplantation, resulted from non-compliance with immunosuppressive therapy.     

Clinical and pathological assessment of acute vascular rejection in the transplant kidney

Koike J, Yamaguchi Y, Horita S, Tanabe K, Fuchinoue S, Toma H, Nihei H. Clinical and pathological assessment of acute vascular rejection in the transplant kidney. Clin Transplantation 2001: 15 (Supplement 5): 41–44. ©Munksgaard, 2001
Acute vascular rejection (AVR) in kidney transplan- tation is the most important factor influencing graft prognosis. We focus on patients whose grafts were lost because of AVR, and assessed their clinical characteristics and histological findings of biopsied renal grafts. Biopsied specimens exhibited AVR in 43 patients who underwent kidney transplantation in the Kidney Center of Tokyo Women's Medical University from 1995 to 1999. In the follow-up from 1 to 5 yr (median: 2.5 yr) we classified these patients into three groups: favourable prog- nosis group (FPG), relatively poor prognosis group (RPPG) and poor prognosis group (PPG). Light microscopic study for histological grading of acute rejection according to the Banff scheme and detection of the C4d complement deposition on peritubular capillaries by the immunofluorescence method were performed. Based on the results, the donors of RPPG and PPG were significantly older than those of FPG, and all factors of acute rejection according to the Banff scheme were not statistically significantly different among the three groups. However, an acute tubular injury mimicking acute tubular necrosis (ATN) was observed in the biopsy specimens from PPG. In conclusion, an older donor is a risk factor of poor prognosis of the graft with AVR, and acute tubular injury mimicking ATN is one of the important features that enables the prediction of graft failure originating from AVR in kidney transplantation.     

Pig islet xenograft rejection is markedly delayed in macrophage-depleted mice: a study in streptozotocin diabetic animals

The present study aimed to evaluate the effect of depletion of macrophages and/or natural killer (NK) cells on islet xenograft rejection in the pig-to-mouse model. Five microliters (4,000 to 5,000 IEQ, islet equivalents) of adult pig islets were transplanted under the renal capsule of C57BL/6 mice with streptozotocin-induced diabetes. Macrophages were depleted by injection of liposome-encapsulated dichloromethylene diphosphonate (Lip-Cl2MDP) intraperitoneally (i.p.) at a dose of 100 microl/ 10 g body weight (BW) 2 days before transplantation, and 50 microl/10 g BW weekly thereafter. NK cells were depleted by injection of the monoclonal antibody NK 1.1 (anti-NK 1.1 mAb) i.p. at a dose of 100 microg/mouse 1 day before transplantation, and then 25 microg per week thereafter. Islet graft survival was monitored by daily measurements of blood glucose. Graft survival was 8 +/- 1.2 days in untreated controls, 9 +/- 1.0 days with anti-NK 1.1 mAb alone, 22 +/- 4.9 days with Lip-Cl2MDP alone (P<0.01 vs. controls), and 26 +/- 3.8 days with Lip-Cl2MDP plus anti-NK 1.1 mAb (P<0.01 vs. controls). In the last group, two of six animals were killed with functioning grafts 30 days after transplantation. In untreated controls, rejected xenografts were heavily infiltrated by F4/80+ macrophages and CD3+T cells. In Lip-Cl2MDP-treated groups, the number of F4/80+ macrophages was markedly reduced. On the periphery of xenografts, a small number of CD3+T cells were observed. In conclusion, our results suggest that strategies targeting macrophages may facilitate islet xenograft survival. A role for NK cells cannot be excluded, but appears to be of minor importance.     

Pig islet xenograft rejection in a mouse model with an established human immune system

Abstract: Background: Xenotransplantation from pigs provides a potential solution to the severe shortage of human pancreata, but strong immunological rejection prevents its clinical application. A better understanding of the human immune response to pig islets would help develop effective strategies for preventing graft rejection. Methods: We assessed pig islet rejection by human immune cells in humanized mice with a functional human immune system. Humanized mice were prepared by transplantation of human fetal thymus/liver tissues and CD34⁺ fetal liver cells into immunodeficient mice. Islet xenograft survival/rejection was determined by histological analysis of the grafts and measurement of porcine C‐peptide in the sera of the recipients. Results: In untreated humanized mice, adult pig islets were completely rejected by 4 weeks. These mice showed no detectable porcine C‐peptide in the sera, and severe intra‐graft infiltration by human T cells, macrophages, and B cells, as well as deposition of human antibodies. Pig islet rejection was prevented by human T‐cell depletion prior to islet xenotransplantation. Islet xenografts harvested from T‐cell‐depleted humanized mice were functional, and showed no human cell infiltration or antibody deposition. Conclusions: Pig islet rejection in humanized mice is largely T‐cell‐dependent, which is consistent with previous observations in non‐human primates. These humanized mice provide a useful model for the study of human xenoimmune responses in vivo.     

Improved suppression of circulating complement does not block acute vascular rejection of pig-to-rhesus monkey cardiac transplants

At present, acute vascular rejection (AVR) remains a primary obstacle inhibiting long-term graft survival in the pig-to-non-human primate transplant model. The present study was undertaken to determine whether repetitive injection of low dose Yunnan-cobra venom factor (Y-CVF), a potent complement inhibitor derived from the venom of Naja kaouthia can completely abrogate hemolytic complement activity and subsequently improve the results in a pig-to-rhesus monkey heterotopic heart transplant model. Nine adult rhesus monkeys received a heterotopic heart transplant from wild-type pigs and the recipients were allocated into two groups: group 1 (n = 4) received repetitive injection of low dose Y-CVF until the end of the study and group 2 (n = 5) did not receive Y-CVF. All recipients were treated with cyclosporine A (CsA), cyclophosphamide (CyP) and steroids. Repetitive Y-CVF treatment led to very dramatic fall in CH50 and serum C3 levels (CH50 < 3 units/C3 remained undetectable throughout the experiment) and successfully prevented hyperacute rejection (HAR), while three of five animals in group 2 underwent HAR. However, the continuous suppression of circulating complement did not prevent AVR and the grafts in group 1 survived from 8 to 13 days. Despite undetectable C3 in circulating blood, C3 deposition was present in these grafts. The venular thrombosis was the predominant histopathologic feature of AVR. We conclude that repetitive injection of low dose Y-CVF can be used to continuously suppress circulating complement in a very potent manner and successfully prevent HAR. However, this therapy did not inhibit complement deposition in the graft and failed to prevent AVR. These data suggest that using alternative pig donors [i.e. human decay accelerating factor (hDAF)-transgenic] in combination with the systemic use of complement inhibitors may be necessary to further control complement activation and improve survival in pig-to-non-human primate xenotransplant model.     

Aggregation of human platelets induced by porcine endothelial cells is dependent upon both activation of complement and thrombin generation

Abstract: The binding of human xenoreactive antibody (XNA) to porcine endothelium with complement (C) activation via the classical pathways is considered the major event triggering hyperacute rejection (HAR) with microvascular thrombosis in vivo. As C components are linked to key events in blood coagulation, we have examined pathways whereby activation of complement by endothelial cells results in xenogeneic platelet activation in vitro. Methods: Cultured porcine aortic endothelial cells (pEC), human aortic endothelial cells (HAEC) or human umbilical vein endothelial cells (HUVEC) were prepared in suspension (5 × 10⁶/ml) using EDTA-collagenase. Human platelet rich plasma (PRP) and washed platelets with platelet poor plasma (PPP) were prepared from control, drug free, volunteer donors. All aggregation tests used a two-sample, four-channel, model 560 Ca Lumi-Aggregometer (Chronolog Corporation, Havertown, PA). Selected assays for complement (C3a; C5b-C9; CH50; and AP50) were then performed on supernatant fluids. To test the effects of complement inhibition and thrombin antagonists, the following agents were pre-incubated with PRP (or PPP) in various titrations for 10 min at 37°C prior to combination with pEC in the aggregometer: soluble complement receptor typel (sCR1); Cobra Venom Factor (CVF), heparin, hirudin, and anti-CD31 (anti-PECAM). In addition, pEC, HAEC, and HUVEC were incubated with 10–20% human PPP; supernatant fluids were harvested at various time points and used for platelet activation assays and for functional tests of thrombin or levels of thrombin-antithrombin complexes (TAT). Results: pEC but not HAEC/HUVEC resulted in activation of PRP or washed platelets only in the presence of supplemental PPP. Platelet activation could be inhibited by pre-incubation of samples with CVF (2–5 IU/ml to deplete complement components) and to a variable extent with sCRl (1–2 mg/ml). Complement assays confirmed activation of C3 by the classical pathway with reduction of the CH50 while C6 deficient samples also supported platelet activation. Aggregation of platelets was inhibited by preincubation of PRP with concentrations of hirudin (1 unit/ml) and heparin (5–10 units/ml) sufficient to neutralize thrombin generated. Supernatant fluids containing PPP removed from pEC were found to have increased levels of thrombin and thrombin-antithrombin complexes and could also activate platelets in a hirudin sensitive manner. Discussion: Platelet and pEC combinations underwent aggregation only in the presence of complement components. This process appeared independent, at least in part, of the assembly of terminal complement components. Consequent pEC generation of thrombin appeared adequate to trigger platelet aggregation which could in turn be inhibited by hirudin or heparin in vitro.     

Hyperacute lung rejection in the pig-to-human model. 2. Synergy between soluble and membrane complement inhibition

Azimzadeh A, Zorn GL III, Blair KSA, Zhang JP, Pfeiffer S, Harrison RA, Cozzi E, White DJG, Pierson RN III. Hyperacute lung rejection in the pig‐to‐human model. 2. Synergy between soluble and membrane complement inhibition. Xenotransplantation 2003; 10: 120–131. © Blackwell Munksgaard, 2003 Background. The role of complement in hyperacute lung xenograft rejection has not been elucidated. The present study evaluates the effect of complement (C) C3/C5 convertase inhibition on hyperacute rejection of pig lung by human blood. Methods. In an established ex‐vivo model, lungs from pigs heterozygous for human decay accelerating factor (hDAF), non‐transgenic littermate control pigs, or farm‐bred pigs were perfused with fresh human blood that was either unmodified or treated with soluble complement receptor type 1 (sCR1: TP10, 100 μg/ml). Results. Non‐transgenic lungs from littermate controls had a median survival time of 35 min (range 5 to 210; P=0.25 vs. farm‐bred piglets: median 5 min, range 5 to 10). Lungs expressing hDAF survived for a median of 90 min (range 10 to 161; P=0.5 and 0.01 vs. littermate and farm‐bred controls, respectively), with sCR1, whereas hDAF (–) lungs failed by 35 min (range 6 to 307), hDAF (+) lungs survived for 330 min (range 39 to 577) [P=0.002 vs. farm‐bred; P=0.08 vs. hDAF (–); P=0.17 vs. sCR1/hDAF (–)]. The rise in pulmonary vascular resistance (PVR) at 5 min was blunted only by hDAF (+) with sCR1 (0.26 ± 0.2 vs. 0.5 to 0.7 mmHg/ml/min for other groups). Plasma C3a and sC5b‐9 and tissue deposition of C5b‐9 were dramatically diminished using sCR1, and further decreased in association with hDAF. Histamine and thromboxane were produced rapidly in all groups. Conclusion. Complement plays an important role in lung HAR. However, even potent inhibition of C3/C5 convertase, both membrane bound in lung and by a soluble‐phase inhibitor in the blood, does not prevent activation of inflammatory responses known to be particularly injurious to the lung. Our findings implicate a role for innate immune pathways resistant to efficient complement regulation. The role of anti‐species antibody, coagulation pathway dysregulation, and additional environmental or genetic influences remain to be defined.     

A rare case of vascular rejection in a renal transplant recipient with nephrotic range proteinuria

Abstract:  In the post-cyclosporine A era, it has been reported that acute rejection after kidney transplantation is commonly revealed as an asymptomatic increase in the serum creatinine level. Nephrotic range proteinuria is observed in patients with recurrent or de novo glomerulonephritis, or with chronic transplant nephropathy and glomerulopathy in the late phase. Acute rejection occurring with nephrotic range proteinuria without a rise of serum creatinine has been rarely reported. Here, we report a rare case of vascular rejection in a renal transplant recipient with nephrotic range proteinuria. A 34-yr-old male renal transplant recipient presented with acute vascular rejection and early-onset nephrotic syndrome. Severe nephrotic range proteinuria was detected with a minimally elevated level of serum creatinine. Biopsy showed severe glomerulitis and vasculitis, which was relieved by conversion of the immunosupressant regimen. Severe proteinuria was a sign of acute vascular rejection with severe glomerulitis and vasculitis. Careful observation to ensure maintenance of immunosuppression is necessary in such cases.