Lars Wennberg

ABO-Incompatible Kidney Transplantation Using Antigen-Specific Immunoadsorption and Rituximab: A 3-Year Follow-up

Background. In 2001 a protocol for ABO-incompatible (ABOi) kidney transplantation based on antigen-specific immunoadsorption and rituximab was introduced at our center, short-term results being comparable with those of ABO-compatible (ABOc) living donor kidney transplantation. Of greater importance, however, is long-term graft function, thus far not evaluated. The aim of this study was therefore to assess long-term results of this protocol. Methods. Twenty ABOi kidney recipients with more than 12-month follow-up were included in the study: all adult crossmatch negative ABOi kidney recipients (n=15) were compared with an adult ABOc living donor recipient control group (n=30), and all pediatric ABOi kidney recipients (<16 years of age) (n=5) were compared with a group of pediatric ABOc kidney recipients (n=18). Results. Mean follow-up was three years. There was no significant difference in patient survival, nor in graft survival or in the incidence of acute rejection in any of the groups. In the adult kidney recipients mean glomerular filtration rate was equivalent at all time points (79–83 mL/min), as was &Dgr;s-creatinine. In the pediatric groups, &Dgr;s-creatinine was similar but glomerular filtration rate lower among the ABOi kidney recipients. There was a significant reduction (P<0.0001) without rebound in A/B antibody titers after transplantation (median IgG 1:2 and median IgM 1:1>1 year posttransplant) compared with pretransplant levels (median IgG 1:32 and IgM 1:16). Conclusion. We conclude that ABOi kidney transplantation using antigen-specific immunoadsorption and rituximab is equivalent to ABOc living donor kidney transplantation. ABOi transplantation after this protocol does not have a negative impact on long-term graft function.

Dissecting the instant blood-mediated inflammatory reaction in islet xenotransplantation.

Abstract:  Background:  A massive destruction of transplanted tissue occurs immediately following transplantation of pancreatic islets from pig to non‐human primates. The detrimental instant blood‐mediated inflammatory reaction (IBMIR), triggered by the porcine islets, is a likely explanation for this tissue loss. This reaction may also be responsible for mediating an adaptive immune response in the recipient that requires a heavy immunosuppressive regimen.

Fetal porcine islet-like cell clusters transplanted to cynomolgus monkeys: an immunohistochemical study.

BACKGROUND The mechanism(s) involved in acute cellular xenograft rejection have hitherto been generated in vitro or in different experimental models, with pig tissue being transplanted to rodents. There is an urgent need to validate these results in a clinically more relevant combination of species. METHODS Fetal porcine islet-like cell clusters (ICC) were transplanted under the kidney capsule in cynomolgus monkeys, either untreated or given immunosuppression with cyclosporine (CsA; 10 mg/kg body weight, intramuscularly) and 15-deoxyspergualin (DSG; 5 mg/kg body weight, intramuscularly). ICC xenografts were examined at 1, 3, 6, or 10-12 days after transplantation, using immunohistochemical techniques. Serum levels of xenoreactive antibodies were measured with ELISA. RESULTS No deposits of IgM, IgG, Clq, or C3 were detected within the ICC xenograft in any of the monkeys. Likewise, no significant increase in the levels of xenoreactive antibodies were found after transplantation. In untreated animals, a few N-Elastase-positive cells (neutrophil granulocytes) were seen in the xenograft at day 1. A few mononuclear cells were present in the adjacent renal parenchyma, but they did not infiltrate the xenograft. At this time (day 1), early signs of necrosis were observed in the central parts of the graft. On day 3, the graft had a large, central necrotic area that contained polymorphonuclear cells; the remaining parts of the xenograft showed severe infiltration with CD8+ T cells. Occasional CD68+ cells (macrophages) were seen on days 1 and 3. On day 6, large numbers of macrophages were found infiltrating the entire graft. A few CD20+ B cells, accumulated as small clusters, were also found. Only a few natural killer cells (CD56+) were detected. The CsA/DSG-treated monkeys showed markedly fewer CD2+/CD8+ T cells on day 6 than the untreated monkeys, and the ICC graft was clearly better preserved. However, the number of CD8+ and CD68+ cells had increased considerably at 12 days after transplantation and diffusely infiltrated the whole ICC xenograft. CONCLUSION Porcine ICC transplanted under the kidney capsule in cynomolgus monkeys were rejected by an acute cell-mediated rejection progressing during the first 6 days after transplantation. The process was not dependent on host Ig or C3 binding to the graft. Although the rejection of porcine ICC was significantly delayed in CsA/DSG-treated monkeys, the ICC xenografts were almost completely destroyed 12 days after transplantation.

Survival of macroencapsulated allogeneic parathyroid tissue one year after transplantation in nonimmunosuppressed humans.

The use of immunoisolation devices may allow transplantation without need for immunosuppression and could widen the indications for cell transplantation. In this study, we evaluated the survival of encapsulated parathyroid tissue in nonimmunosuppressed humans. Autologous parathyroid implants: Seven patients under-going parathyroidectomy had devices containing small pieces of their own parathyroid tissue implanted SC. These devices were explanted after 2–4 weeks for histological evaluation. Allogeneic parathyroid implants: Four patients with chronic hypoparathyroidism were transplanted with one to three large (40 μl) and one small (4.5 μl) device filled with meshed parathyroid tissue and implanted SC. The small devices were explanted at 4 weeks, while the large ones were explanted 8.5 to 14 months after implantation. In both studies, control implants were placed in nude mice. Autologous study results: At explantation, the grafts consisted of 22 ± 6% endocrine tissue and 63 ± 7% fibrosis, while 15 ± 5% of the grafts were necrotic. Allogeneic study results: In devices explanted from the patients at 4 weeks, fibrosis dominated and only 1%, 5%, and 23% of the grafts consisted of endocrine tissue. A similar histological appearance was found in grafts from nude mice. In devices explanted at 8.5–14 months, histologically intact endocrine tissue was found in all patients. However, nearly all the tissue consisted of fibrosis. There was no detectable increase in the parathormone (PTH) level in all patients. Macroencapsulated human allogeneic parathyroid tissue can survive up to 1 year after transplantation into nonimmunosuppressed patients. However, marked fibroblast overgrowth occurred, especially in the allogeneic implant study, using meshed parathyroid tissue. This was probably not related to the allo-response, because similar findings were observed in the nude mouse implants. In future studies, better tissue preparation and improvements in the physiological milieu inside the device may help to reduce fibroblast overgrowth and increase survival of the parathyroid cells.

Diabetic rats transplanted with adult porcine islets and immunosuppressed with cyclosporine A, mycophenolate mofetil, and leflunomide remain normoglycemic for up to 100 days.

Background. Transplantation of adult porcine islets (APIs) offers a possible means of treating diabetes. However, isolating APIs has been notoriously difficult. Furthermore, islet xenograft rejection must be prevented. Materials and Methods. APIs were isolated by a modified automated method. API quality was assessed by static glucose stimulation (SGS), by transplantation to diabetic nude mice and by intraperitoneal glucose tolerance tests (IPGTTs). The morphologic characteristics of API xenograft rejection in rats were studied immunohistochemically. Furthermore, APIs were transplanted to diabetic rats that were either left untreated or immunosuppressed with cyclosporine A (CsA), mycophenolate mofetil (MMF) and leflunomide (LEF). B-glucose and porcine C-peptide levels were monitored and grafts were studied morphologically. Results. Large numbers of APIs were isolated. At SGS, insulin release increased significantly. All nude mice transplanted with APIs were normoglycemic within 24 hr and remained so for up to 1 year. During IPGTTs, B-glucose levels were rapidly regulated to porcine levels. In untreated rats, API xenografts were destroyed within 6 days by a cellular infiltrate consisting mainly of macrophages. In untreated diabetic rats normoglycemia was sustained for 5.5±0.3 days. Rats immunosuppressed with CsA+MMF+LEF remained normoglycemic for 59.6±11.3 days. In 3 of 11 rats, normoglycemia was sustained for up to 101 days. Porcine C-peptide was detected in serum. At recurrence of hyperglycemia, many mononuclear cells were found close to the xenografts. However, only occasional cells infiltrated the grafts and many APIs were intact. Conclusions. Well-functioning APIs can be isolated in large numbers. API xenografts can be protected from rejection and can maintain an adequate function for up to 100 days, in rats immunosuppressed with CsA+MMF+LEF.

Xenoislet transplantation: experimental and clinical aspects.

Ten diabetic renal transplant patients had porcine fetal islet-like cell clusters (ICC) injected intraportally or placed under the kidney capsule. In some patients, temporary graft survival was achieved, as evidenced by the urinary excretion of small amounts of porcine C-peptide (4 patients) and the identification of some intact insulin-staining cells in a biopsy specimen (1 patient). Glucose metabolism remained unaffected. To improve the results, better islets and better immunosuppressive protocols are required. We found that, while fetal porcine ICC produced insulin only after several weeks, adult islets gave immediate insulin production. The search for an optimal immunosuppression was conducted in the pig-to-rat islet transplant model. A clear inhibitory effect on the xenograft rejection was observed when using some of the new drugs. The best results were achieved with a triple drug regimen consisting of cyclosporine, mycophenolate mofetil and leflunomide.

Efficacy of immunosuppressive drugs in islet xenotransplantation: leflunomide in combination with cyclosporine and mycophenolate mofetil prevents islet xenograft rejection in the pig-to-rat model.

BACKGROUND Morphological characteristics of islet xenograft rejection differ from those of islet allograft rejection. Therefore, prevention of islet xenograft rejection probably requires a different type of immunosuppression from that used in allogeneic transplantation. METHODS Fetal porcine islet-like cell clusters (ICC) were transplanted into the renal subcapsular space of rats treated with different immunosuppressive protocols. The existence of a cellular infiltrate or deposits of antibodies and complement in the grafts was evaluated at different times after transplantation using immunohistochemistry. RESULTS Treatment with leflunomide (LEF), cyclosporine (CsA), mycophenolate mofetil (MMF), 15-deoxyspergualin, and rapamycin alone or in combination had an insufficient inhibitory effect on ICC xenograft rejection. However, in animals treated with LEF+CsA, the rejection process was markedly inhibited. However, some macrophages and T cells were still present, and at 24 days, the xenografts were destroyed. In LEF+CsA-treated animals that were given sera containing an excessive amount of rat anti-porcine xenoreactive antibodies, marked deposits of IgG, and to some extent C3 as well, were detected along the border between intact ICC, and the xenografts were surrounded by macrophages. However, almost no cells infiltrated the grafts, and there were many intact ICC. In animals treated with LEF+CsA+MMF, only occasional infiltrating cells were seen at 12 and 24 days after transplantation, and the endocrine tissue was completely intact. CONCLUSIONS LEF+CsA+MMF prevented rejection of porcine ICC xenografts in the rat for up to 24 days after transplantation.

Pharmacodynamics of rituximab in kidney transplantation.

The B-cell depleting anti-CD20 antibody rituximab has become a therapeutic alternative in renal transplantation. However, understanding of the pharmacodynamics is limited. We have therefore studied the effect of single-dose rituximab, in combination with conventional triple immunosuppressive therapy, on the B-cell population in peripheral blood as well as in tissues, in kidney transplant recipients. Forty-nine kidney recipients received single-dose rituximab. The prevalence of B cells was assessed in peripheral blood, kidney transplant tissue, and in lymph nodes. In 88%, complete depletion of B cells in peripheral blood was observed and, 15 months after treatment, B cells were still undetectable in the majority of patients. In kidney tissue, B cells were also completely eliminated. In contrast, the B cells were not eliminated in lymph nodes, although a reduction was observed. In conclusion, single-dose rituximab in kidney transplant recipients evokes a long-term elimination of B-cells in peripheral blood as well as within the kidney transplant.

Effects of immunosuppressive treatment on host responses against intracerebral porcine neural tissue xenografts in rats

Background. Embryonic xenogeneic neural tissue is an alternative for transplantation in Parkinson’s disease, but immune responses limit the application. The aims of this study were to enhance the in vitro viability rates by donor tissue pretreatment; to compare the efficacy of cyclosporine A (CsA) and tacrolimus (FK) in inhibiting xenograft rejection in rats; to evaluate additional inductive therapy with prednisolone (PRE) or mycophenolate mofetil (MMF). Methods. Tirilazad (a lipid peroxidase inhibitor) or FK and acYVAD-cmk (a caspase inhibitor), were added to embryonic porcine ventral mesencephalic tissue and viability was assessed in vitro. Tirilazad-treated tissue was grafted to the striatum of rats that were either left untreated or immunosuppressed with FK (1 mg/kg) or CsA (15 mg/kg) alone or in combination with a 2-week PRE (20 mg/kg) or MMF (40 mg/kg) induction course. Xenograft survival and host responses were determined using immunohistochemistry. Results. Pretreatment with tirilazad enhanced tissue survival in vitro. After transplantation into untreated controls, there was no graft survival at twelve weeks. Neural cell counts were significantly improved in immunosuppressed recipients, but there were no differences between the treatment groups. Additional inductive treatment reduced the infiltration with CD4+ and CD8+ cells, and macrophage infiltration was reduced compared with animals given CsA or FK alone. Conclusion. Pretreatment of the donor tissue with free-radical scavengers reduces cell loss caused by tissue trauma. Porcine neural tissue xenografts survive significantly better in animals immunosuppressed with either FK or CsA. Additional inductive treatment with PRE or MMF reduced the infiltration of host cells into the xenografts.

The efficacy of antigen-specific immunoadsorption and rebound of anti-A/B antibodies in ABO-incompatible kidney transplantation

BACKGROUND As antigen-specific immunoadsorption (IA) using the Glycosorb®-ABO columns is becoming increasingly popular in ABO-incompatible (ABOi) transplantation, in this study, we retrospectively investigated the efficacy of Glycosorb®-ABO IA in vivo and ex vivo. We also assessed the risk of anti-A/B antibody (ABab) rebound before and after ABOi kidney transplantation. METHODS A protocol for ABOi living donor kidney transplantation was used, combining four preoperative and three preemptive postoperative Glycosorb®-ABO IAs with rituximab and maintenance immunosuppression. ABabs were determined by a haemagglutination titration technique. RESULTS ABOi kidney transplantation was attempted 45 times and 43 transplantations were performed. Overall patient survival was 93% and graft survival was 91%. Mean follow-up was 4.5 years. Glycosorb®-ABO IA significantly reduced the ABabs in the majority of patients (P < 0.0001). However, in three patients (6.8%), the antibody elimination was incomplete. Inadequate adsorption of core-chain-dependent ABabs may explain this finding, but further studies are needed. In five patients, the preconditioning was interrupted before transplantation, resulting in ABab rebound. Yet, when preconditioning was restarted, the antibodies could be removed as planned. After ABOi transplantation, rebound of ABabs was seen in two patients (5%). CONCLUSIONS Glycosorb®-ABO IA in combination with rituximab effectively depletes ABabs in most patients, but owing to core-chain-dependent ABabs, Glycosorb®-ABO IA may be less effective than nonspecific techniques for antibody removal in some patients. Rebound before transplantation subsequent to interrupted preconditioning does not hamper a successful ABOi transplantation. Postoperatively, when this protocol for ABOi transplantation is followed, the risk of ABab rebound is small.