IgE-Mediated Immune Response and Antibody-Mediated Rejection.

BACKGROUND AND OBJECTIVES: Active antibody-mediated rejection is the main cause of kidney transplant loss, sharing with SLE the alloimmune response and the systemic activation of the IFN-α pathway. IgE-mediated immune response plays a key role in the development of SLE nephritis and is associated with IFN-α secretion. The aim of our study was to investigate IgE-mediated immune response in antibody-mediated rejection. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: This was a cross-sectional study of 56 biopsy-proven antibody-mediated rejection study participants, 80 recipients with normal graft function/histology (control), 16 study participants with interstitial fibrosis/tubular atrophy, and six participants with SLE. We evaluated graft IgE deposition, tryptase (a mast cell marker), and CD203 (a specific marker of activated basophils) by immunofluorescence/confocal microscopy. In addition, we measured serum concentration of human myxovirus resistance protein 1, an IFN-α-induced protein, and anti-HLA IgE. RESULTS: We observed a significantly higher IgE deposition in tubules and glomeruli in antibody-mediated rejection (1766±79 pixels) and SLE (1495±43 pixels) compared with interstitial fibrosis/tubular atrophy (582±122 pixels) and control (253±50 pixels). Patients with antibody-mediated rejection, but not control patients and patients with interstitial fibrosis/tubular atrophy, presented circulating anti-HLA IgE antibodies, although with a low mean fluorescence intensity. In addition, immunofluorescence revealed the presence of both mast cells and activated basophils in antibody-mediated rejection but not in control and interstitial fibrosis/tubular atrophy. The concentration of circulating basophils was significantly higher in antibody-mediated rejection compared with control and interstitial fibrosis/tubular atrophy. MxA serum levels were significantly higher in antibody-mediated rejection compared with control and correlated with the extent of IgE deposition. CONCLUSIONS: Our data suggest that IgE deposition and the subsequent recruitment of basophils and mast cells within the kidney transplant might play a role in antibody-mediated rejection.

Glomerulitis and endothelial cell enlargement in C4d+ and C4d- acute rejections of renal transplant patients.

In acute rejection after renal transplant, glomerulitis is characterized by mononuclear cells in glomerular capillaries and endothelial cell enlargement. In association with C4d deposition in peritubular capillaries, glomerulitis is a feature of acute antibody-mediated rejection. Prognosis in C4d(+) rejection is poorer than in C4d(-) rejection. We measured the glomerular endothelial cell area in C4d(+) and C4d(-) acute rejections by morphometry. In 90 acute rejection biopsies, glomerulitis was present in 36 cases (group G) and absent in 54 (group G0). In biopsies without rejections and in C4d(-) biopsies of group G0, glomerular endothelial cell area was not significantly different. In C4d(-) and C4d(+) biopsies of group G, the area in inflamed glomeruli was greater than that in C4d(-) biopsies of group G0 (P < .02 and P < .006, respectively). In C4d(+) biopsies of group G0, it was, unexpectedly, greater than in C4d(-) biopsies of group G (P < .01). Circulating posttransplant anti-human leukocyte antigen class I and class II antibodies correlated with increased endothelial cell area (P < .02). Glomerulitis was associated with diffuse C4d deposition (odds ratio [OR], 4.27; P < .004); C4d deposition was associated with steroid resistance (OR, 4.97; P < .002). Only in C4d(+) rejections did the presence of glomerulitis increase this association (OR, 9.17; P < .02). In conclusion, we quantified an increase of endothelial cell area in glomerulitis of C4d(+) and C4d(-) acute rejections (group G). An increase of this area in C4d(+) biopsies without glomerulitis (group G0) suggests complement-mediated damage in the absence of mononuclear cell margination.

[Role of antibodies in kidney transplant]. / Ruolo degli anticorpi nel trapianto di rene.

Rejection is one of the most frequent causes of graft loss after a kidney transplant. In this context, in the last few years the essential role of antibodies in the anti-graft immune response has become more evident. Antibody-mediated damage has been classified into four histological patterns: hyperacute rejection, caused by the presence of pre-existing donor-specific antibodies directed against HLA or non-HLA antigens; acute antibody-mediated rejection, usually due to antibodies elicited following transplantation; chronic antibody-mediated rejection, which can develop months or years after the first appearance of circulating antibodies; and Cd4 deposition without morphologic evidence of active rejection, previously described as ''accommodation''. In recent years, thanks to the development of specific desensitization protocols, it has become possible to transplant patients sensitized to donor HLA antigens. Recently, besides consolidated protocols which include immunoglobulin administration associated or not with plasmapheresis, novel approaches of therapeutic apheresis with specific removal of antibodies and bortezomib, an agent that can efficiently decrease donor-specific antibody levels, have been developed. As far as the treatment of antibody-mediated rejection is concerned, different immunosuppressive strategies have been used. These include the combination of immunoglobulin administration and plasmapheresis with or without the use of an anti-CD20 monoclonal antibody. More recently, an innovative therapy with eculizumab has proved to be very effective against acute antibody-mediated rejection. The debate regarding the cause-effect relationship between the development of an early post-transplant humoral immune response in patients with stable graft function and premature graft loss remains open to discussion. Clinical studies are underway to provide an adequate answer to this question. In conclusion, comprehension of the fundamental role of antibodies and the consolidation of desensitization techniques together with early treatment of antibody-mediated rejection remain important objectives to improve long-term allograft survival.

Effects of long-term administration of recombinant human protein C in xenografted primates.

BACKGROUND: The role potential of recombinant human activated protein C (rhaPC), a recently developed molecule with anticoagulant and antiinflammatory properties, in prolonging survival in immunosuppressed primate recipients of porcine renal xenografts has been evaluated. METHODS: rhaPC was administered daily for 5 days (24 µg/kg/hr; group A; n = 3) or throughout the postoperative period (8-24 µg/kg/hr; group B; n = 2; or 24-48 µg/kg/hr; group C; n = 4). Animals in group D (n = 2) received rhaPC daily (24 µg/kg/hr) combined with recombinant human antithrombin (84 U/kg every 8 hr). Two animals served as control (group E). RESULTS: The results indicate that rhaPC is protective against fibrin deposition early after transplantation but does not prevent fibrin deposition and the occurrence of acute humoral xenograft rejection (AHXR) later on. Animals in the study survived between 8 and 55 days. At the dose used, rhaPC is able to prevent fibrin deposition in the graft in the first 2 weeks after xenotransplantation, except when it is administered in conjunction with antithrombin. However, rhaPC did not prevent the eventual occurrence of AHXR in primate recipients of porcine xenografts. CONCLUSIONS: In this pig to primate model, rhaPC confers a short advantage in the prevention of early perioperative xenograft damage but does not represent an effective strategy for preventing AHXR.

In vitro and in vivo immunomodulatory effects of cobalt protoporphyrin administered in combination with immunosuppressive drugs.

BACKGROUND: Immunosuppressive strategies are designed to take advantage of potential synergies between drugs to possibly decrease the risk of side-effects. In the present study, the ability of Cobalt protoporphyrin (CoPP) to potentiate the effect of the immunosuppressive drugs mycophenolate sodium (MPS) or cyclosporin A (CsA) was explored in vitro and in vivo. METHODS: In vitro analyses of proliferation and apoptosis were performed on primate T cell cultures, following incubation with the immunosuppressive drugs MPS or CsA, alone or in combination with CoPP. In vivo the effect of CoPP and CsA combination therapy was assessed in a rat heterotopic cardiac allotransplantation model. RESULTS: In vitro results suggest that co-administration of CoPP with CsA or MPS increases immunosuppressive effects of these drugs when combined with CoPP. In particular, the co-administration of CoPP with CsA resulted in the synergistic induction of lymphocyte apoptosis. In vivo, animals immunosuppressed with CsA (1.5 mg/kg) or CoPP (20 mg/kg) alone, had a median survival of 7 or 8 days, respectively. In contrast, animals immunosuppressed with CsA (1.5 mg/kg) combined with CoPP (20 mg/kg) had significantly prolonged median survival (12 days), compared to recipients treated with CsA or CoPP alone (p<0.05). CONCLUSION: Our in vitro and in vivo studies demonstrate that CoPP can potentiate the immunomodulatory effects of CsA, ultimately extending allograft survival.

Xenotransplantation as a model of integrated, multidisciplinary research.

Xenotransplantation was proposed a long time ago as a possible solution to the world-wide shortage of human organs. For years, researchers in this field have almost exclusively directed their efforts towards combating the immunological barrier that precluded long-term xenograft survival. Studies have been conducted in both small and large animal models and the most relevant results have been obtained in pre-clincal studies, specifically those utilising the pig-to-nonhuman primate combination. In this context, a better understanding of the immunological mechanisms underlying the rejection of a xenograft have allowed the identification of specific targets of intervention that have resulted in considerable improvements in survival of porcine organs or cells in nonhuman primates. However it has also become apparent that if xenotransplantation has to enter the clinical arena, a multidisciplinary approach will be needed to comprehensively tackle the different issues related to the use of a xenograft to cure human disease.In this regard, the safety, ethics and regulatory aspects of xenotransplantation are currently being aggressively addressed to enable the initiation of xenotransplantation with a favourable risk/benefit ratio.

In vitro and in vivo effects of the carbon monoxide-releasing molecule, CORM-3, in the xenogeneic pig-to-primate context.

BACKGROUND: Carbon monoxide (CO) interferes with inflammatory and apoptotic processes associated with ischemia-reperfusion injury and graft rejection. Here, the in vitro effects of carbon monoxide releasing molecule-3 (CORM-3), a novel water-soluble carbonyl CO carrier, have been investigated on porcine aortic endothelial cells (PAEC) and primate peripheral blood mononuclear cells (PBMC). Furthermore, the pharmacodynamics and pharmacotolerance of CORM-3 after administration of single and multiple doses in the primate have been assessed in view of its potential application in pig-to-primate xenotransplantation models. METHODS: For in vitro studies, PAEC and primate PBMC were exposed for 24, 48 and 72 h to CORM-3 (20 to 1000 microm) and viability was measured using an MTS assay. PAEC and primate PBMC proliferation after exposure to CORM-3 was assessed by CFSE labelling. Proliferation of primate PBMC against irradiated pig lymphocytes was also assessed. Tumor necrosis factor alpha (TNF-alpha) production and Caspase-3 and -7 activity in Concanavalin A (conA)-stimulated primate PBMC were measured following treatment with CORM-3. In vivo, CORM-3 was administered i.v. to cynomolgus monkeys at 4 mg/kg, as single or multiple doses for up to 30 days. The effect of CORM-3 was evaluated by the assessment of production of TNF-alpha and interleukin 1beta following PBMC stimulation with LPS by species-specific ELISA. Complete hematologic and biochemical analyses were routinely performed in treated primates. RESULTS: At concentrations <500 microm, CORM-3 did not alter the viability of PAEC or primate PBMC cultures in vitro, nor did it induce significant levels of apoptosis or necrosis. Interestingly, at concentrations of 300 and 500 microm, significant PAEC proliferation was observed, whilst concentrations > or =50 microm inhibited conA-activated primate lymphocyte proliferation (IC(50) of 345.8 +/- 51.9 microm) and the primate xenogeneic response against pig PBMC. Such responses were demonstrated to be CO-dependent. In addition, CORM-3 significantly inhibited caspase-3 and -7 activity at concentrations between 200 and 500 microm and caused a significant reduction in TNF-alpha production (IC(50) 332.8 +/- 33.9 microm). In vivo, following the administration of multiple doses, TNF-alpha production was significantly reduced in comparison to pre-treatment responses, with decreased levels maintained throughout the study. Moreover, a slight and transient increase in transaminases and bilirubin was observed in animals exposed to multiple doses of CORM-3. CONCLUSIONS: These studies suggest that CORM-3 has anti-inflammatory and immunomodulatory properties in primates that may result in clinical benefit to allo- and xenografted organs.

De novo anti-HLA antibody responses after renal transplantation: detection and clinical impact.

Numerous retrospective and prospective studies have been conducted to determine the prevalence and significance on long-term graft survival of de novo post-transplant donor-specific antibodies (DSA), directed against both HLA and non-HLA molecules. Moreover, it has been postulated that the development of anti-HLA antibodies may precede the clinical manifestation of chronic rejection, therefore being considered a predictive marker. In this context, the detection of C4d deposition in the failing kidney in patients presenting de novo DSA supports the hypothesis that antibody production and complement deposition could be involved in the pathogenesis of graft failure. Due to the development of more sensitive meth-ods to detect alloantibodies, the number of transplanted patients which show the appearance of DSA at different times following transplantation has increased. Nevertheless, this increased sensitivity has allowed the identification of circulating donor-specific anti-HLA antibodies in many patients with otherwise good graft function. Such findings are worthy of discussion, as it has yet to be determined whether these circulating antibodies can only be considered an early marker of humoral rejection or whether they could play a protective role. The possible relevance of the post-transplant appearance of non-DSA should also be mentioned. This review will focus primarily on de novo anti-donor HLA antibody responses in kidney transplant patients and will only briefly deal with anti-non HLA and non-DSA that will be discussed elsewhere in this issue.

Transgene expression of green fluorescent protein and germ line transmission in cloned pigs derived from in vitro transfected adult fibroblasts.

The pig represents the xenogeneic donor of choice for future organ transplantation in humans for anatomical and physiological reasons. However, to bypass several immunological barriers, strong and stable human genes expression must occur in the pig's organs. In this study we created transgenic pigs using in vitro transfection of cultured cells combined with somatic cell nuclear transfer (SCNT) to evaluate the ubiquitous transgene expression driven by pCAGGS vector in presence of different selectors. pCAGGS confirmed to be a very effective vector for ubiquitous transgene expression, irrespective of the selector that was used. Green fluorescent protein (GFP) expression observed in transfected fibroblasts was also maintained after nuclear transfer, through pre- and postimplantation development, at birth and during adulthood. Germ line transmission without silencing of the transgene was demonstrated. The ubiquitous expression of GFP was clearly confirmed in several tissues including endothelial cells, thus making it a suitable vector for the expression of multiple genes relevant to xenotransplantation where tissue specificity is not required. Finally cotransfection of green and red fluorescence protein transgenes was performed in fibroblasts and after nuclear transfer blastocysts expressing both fluorescent proteins were obtained.

Cobalt protoporpyhrin reduces caspase-3,-7 enzyme activity in neonatal porcine islets, but does not inhibit cell death induced by TNF-alpha.

Apoptotic phenomena observed in vitro following isolation and following transplantation contribute significantly to islet graft loss. Strategies to reduce apoptosis of islet tissue prior to and posttransplantation may improve graft survival and function and reduce the amount of tissue necessary to achieve insulin independence. The expression of cytoprotective proteins is one such strategy that may prolong islet survival. In this light, heme-oxygenase 1 (HO-1) upregulation has been studied in both allo- and xenotransplantation models. In this study, the effect of HO-1 on apoptosis in neonatal porcine islet-like cell clusters (NPICC) was assessed. In in vitro assessments of NPICC apoptosis, NPICC showed a high sensitivity to apoptotic stimulation using a combination of TNF-alpha and cycloheximide. Stimulation with TNF-alpha alone was sufficient to induce reproducible apoptotic responses as demonstrated by caspase-3,-7 activation and subdiploid DNA analysis. Dose-dependent, high-level HO-1 protein expression was achieved following culture of NPICC in medium containing either cobalt protoporphyrin (CoPP) or cobalt mesoporphyrin (CoMP). CoPP treatment resulted in the reduction of caspase-3,-7 enzyme activity following TNF-alpha stimulation. However, such an effect was not associated with a reduction in the levels of cell death. Indeed, the inhibition of caspase enzyme activity resulted in decreased PARP-1 cleavage, which may lead to heightened levels of necrosis in treated NPICC cultures, possibly explaining the observed commitment of NPICC to the death pathway.

Effects of long-term administration of high-dose recombinant human antithrombin in immunosuppressed primate recipients of porcine xenografts.

BACKGROUND: Fibrin deposition is central to the acute humoral rejection process occurring in the presence of consumptive coagulopathy when pig organs are transplanted into primates. METHODS: To assess whether strategies aimed at preventing fibrin formation may extend xenograft survival, we administered high daily doses of recombinant human antithrombin (rhAT) (500 U/kg twice daily) to obtain both anticoagulant and anti-inflammatory effects in immunosuppressed primate recipients of porcine kidneys. RESULTS: Some degree of consumptive coagulopathy developed in both rhAT-treated (n=3) and untreated (n=3) primates. No major differences in the coagulation parameters analyzed were observed between the 2 groups. Similarly, no difference in survival was seen between rhAT-treated (20.6+/-4 days; range: 15-23 days) and untreated animals (17.3+/-11.6 days; range: 7-30 days), although the rhAT-treated primates had a higher bleeding tendency. Despite the high daily dose of rhAT, considerable fibrin deposition was observed in the graft as early as 2 weeks after transplantation. CONCLUSIONS: These results suggest that a high daily dose of rhAT fails to influence survival or prevent fibrin formation and deposition in the graft in our pig-to-primate model. However, the potential role of rhAT administered in combination with heparins or other clotting inhibitor concentrates in this model remains to be determined.

Host-derived circulating cells do not significantly contribute to cardiac regeneration in heterotopic rat heart transplants.

OBJECTIVES: The aim of this study was to investigate the contribution of host-derived circulating cells to cardiac repair after tissue damage using the model of heterotopic heart transplantation between transgenic recipient rats expressing green fluorescent protein (GFP) and wild-type donors. METHODS: Unlabeled donor rat hearts, some of which underwent prolonged cold ischemia pretreatment, were transplanted into the abdominal cavity of GFP+ transgenic recipient rats and were analyzed 15 and 90 days after surgery. An additional experimental group underwent heart transplantation following administration of granulocyte-colony stimulatory factor (G-CSF) to mobilize bone marrow cells. RESULTS: Most transplants contained GFP+ mature cardiomyocytes. However, systematic counting in the transplants showed that the proportion of GFP+ cardiomyocytes was only 0.0005% to 0.008% of all cardiomyocytes. These relative proportions did not change after G-CSF treatment, despite evidence for sustained marrow cell mobilization. Confocal image analysis showed that the majority of GFP+ cardiomyocytes contained a high number of nuclei, suggesting that these cells may derive from fusion events. Very rarely, small GFP+ undifferentiated cells, expressing GATA-4, were also identified. Occasionally, GFP+ endothelial cells, but not smooth muscle cells, were detected in blood vessels of some transplants. CONCLUSIONS: Our results demonstrate that cardiomyocytes expressing a host transgenic marker are detectable in heterotopic heart transplants; however, they do not significantly contribute to repopulation of the damaged myocardium.

Xenotransplantation-current status and future perspectives.

Research efforts have shed light on the immunological obstacles to long-term survival of pig organs transplanted into primates and allowed the identification of targets for specific immune intervention. Accordingly, the development of genetically engineered animals has overcome the hyperacute rejection barrier, with acute humoral xenograft rejection (AHXR) currently remaining the most important immunological obstacle. At this stage, a better control of the elicited anti-pig humoral immune response and avoidance of coagulation disorders are the two primary research fronts being pursued in order to overcome AHXR. Nonetheless, it is encouraging that porcine xenografts can sustain the life of non-human primates for several months. Proactive research aimed at the development of a safer organ source is also underway. It is anticipated that ongoing research in several fields, including accommodation, tolerance, immune suppression and genetic engineering, will result in further improvements in non-human primate survival. However, until convincing efficacy data and a more favourable risk/benefit ratio can be established in relevant animal models, progression to the clinic should not be viewed as an option.

Ureteral stenosis in HDAF pig-to-primate renal xenotransplantation: a phenomenon related to immunological events?

The aim of this study was to analyze the incidence of ureteral stenosis in a life-supporting human decay-accelerating factor (hDAF) transgenic pig-to-cynomolgus monkey kidney transplantation model and determine the role of possible immunological events in its pathogenesis. Thirty consecutive bi-nephrectomized cynomolgus monkeys received a kidney from hDAF transgenic pigs with or without a ureteral stent. Four monkeys were euthanized prematurely after transplantation. In the remaining 26 cases, the mean survival was 24 +/- 19 days. Except in one case, there was a close relationship between ureter and kidney in terms of type and severity of rejection. There were six ureteral stenoses; five were repaired by stent positioning and resurgery extended survival for an additional 16 +/- 10 days. The stenotic ureters showed diffuse acute humoral xenograft rejection (AHXR), while all cases with no or only focal signs of ureteral rejection never revealed ureteral obstruction. Use of a ureteral stent extends the survival of a xenografted primate, thereby helping to clarify the immunological events surrounding the onset of AHXR in kidneys in long-term xenograft recipients.

Alterations in the coagulation profile in renal pig-to-monkey xenotransplantation.

Five monkey recipients of a porcine renal xenograft were studied to determine the relationship between fibrin formation in acute humoral xenograft rejection (AHXR) and procoagulant and anticoagulant factor levels to establish whether changes in coagulation parameters could be used to predict AHXR and determine whether AHXR is associated with overt disseminated intravascular coagulopathy (DIC) in this model. Variable degrees of compensated consumptive coagulopathy were observed in each primate. Elevated thrombin-antithrombin (TAT), F1+2 and D-dimer levels consistent with thrombin generation and fibrin formation were recorded. There was no consumption of the main clotting inhibitors (including antithrombin) or a progressive, severe drop in fibrinogen levels and platelet counts, although grafts were left in situ. After transplantation, D-dimer levels remained persistently high, so they were of limited value in defining this coagulopathy. At post mortem, no cases of multiorgan involvement typical of overt DIC were observed. The lack of a rapid postoperative recovery of clotting inhibitor levels after transplantation was invariably associated with early poor outcome. This study shows that AHXR is associated with various degrees of compensated consumptive coagulopathy in our pig-to-primate model. No clear relationship was found between coagulation parameter levels and graft outcome.

Structure of the neutrophil-activating protein from Helicobacter pylori.

Helicobacter pylori is a major human pathogen associated with severe gastroduodenal diseases, including ulcers and cancers. An H.pylori protein that is highly immunogenic in humans and mice has been identified recently. This protein has been termed HP-NAP, due to its ability of activating neutrophils. In order to achieve a molecular understanding of its unique immunogenic and pro-inflammatory properties, we have determined its three-dimensional structure. Its quaternary structure is similar to that of the dodecameric bacterial ferritins (Dps-like family), but it has a different surface potential charge distribution. This is due to the presence of a large number of positively charged residues, which could well account for its unique ability in activating human leukocytes.

Screening inhibitors of anthrax lethal factor.

The disease anthrax is caused by lethal factor, an enzyme component of the toxin produced by the spore-forming bacterium Bacillus anthracis. Here we describe substrate molecules for this factor that offer a means for high-throughput screening of potential inhibitors for use in anthrax treatment. Our assay should help to answer the urgent call for new and specific therapies to combat this pathogen after its recent emergence as a terrorist bioweapon.