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Molecular testing represents a promising adjunct for the diagnosis of antibody-mediated rejection (AMR). Here, we apply a novel gene expression platform in sequential formalin-fixed paraffin-embedded samples from nonhuman primate (NHP) renal transplants. We analyzed 34 previously described gene transcripts related to AMR in humans in 197 archival NHP samples, including 102 from recipients that developed chronic AMR, 80 from recipients without AMR, and 15 normal native nephrectomies. Three endothelial genes (VWF, DARC, and CAV1), derived from 10-fold cross-validation receiver operating characteristic curve analysis, demonstrated excellent discrimination between AMR and non-AMR samples (area under the curve = 0.92). This three-gene set correlated with classic features of AMR, including glomerulitis, capillaritis, glomerulopathy, C4d deposition, and DSAs (r = 0.39-0.63, p < 0.001). Principal component analysis confirmed the association between three-gene set expression and AMR and highlighted the ambiguity of v lesions and ptc lesions between AMR and T cell-mediated rejection (TCMR). Elevated three-gene set expression corresponded with the development of immunopathological evidence of rejection and often preceded it. Many recipients demonstrated mixed AMR and TCMR, suggesting that this represents the natural pattern of rejection. These data provide NHP animal model validation of recent updates to the Banff classification including the assessment of molecular markers for diagnosing AMR.

Summary Induction of allograft tolerance has been considered the ultimate goal in organ transplantation. Although numerous protocols to induce allograft tolerance have been reported in mice, a chimerism-based approach through donor haematopoietic stem cell transplantation has been the only approach to date that induced allograft tolerance reproducibly following kidney transplantation in man. Renal allograft tolerance has been achieved by induction of either transient mixed chimerism or persistent full donor chimerism. Although the risk of rejection may be low in tolerance achieved via durable full donor chimerism, the development of graft-versus-host disease (GVHD) has limited the wider clinical application of this approach. In contrast, tolerance induced by transient mixed chimerism has not been associated with GVHD, but the risk of allograft rejection is more difficult to predict after the disappearance of haematopoietic chimerism. Current efforts are directed towards the development of more clinically feasible and reliable approaches to induce more durable mixed chimerism in order to widen the clinical applicability of these treatment regimens.

The lack of a reliable immunosuppressive regimen that effectively suppresses both renal and islet allograft rejection without islet toxicity hampers a wider clinical application of simultaneous islet-kidney transplantation (SIK). Seven MHC-mismatched SIKs were performed in diabetic cynomolgus monkeys. Two recipients received rabbit antithymocyte globulin (ATG) induction followed by daily tacrolimus and rapamycin (ATG/Tac/Rapa), and five recipients were treated with anti-CD40 monoclonal antibody (mAb) and rapamycin (aCD40/Rapa). Anti-inflammatory therapy, including anti-interleukin-6 receptor mAb and anti-tumor necrosis factor-α mAb, was given in both groups. The ATG/Tac/Rapa recipients failed to achieve long-term islet allograft survival (19 and 26 days) due to poor islet engraftment and cytomegalovirus pneumonia. In contrast, the aCD40/Rapa regimen provided long-term islet and kidney allograft survival (90, 94, >120, >120, and >120 days), with only one recipient developing evidence of allograft rejection. The aCD40/Rapa regimen was also tested in four kidney-alone transplant recipients. All four recipients achieved long-term renal allograft survival (100% at day 120), which was superior to renal allograft survival (62.9% at day 120) with triple immunosuppressive regimen (tacrolimus, mycophenolate mofetil, and steroids). The combination of anti-CD40 mAb and rapamycin is an effective and nontoxic immunosuppressive regimen that uses only clinically available agents for kidney and islet recipients.

BACKGROUND Although induction of durable mixed chimerism is required for murine skin allograft tolerance (TOL), renal allograft TOL has been achieved after induction of only transient mixed chimerism in nonhuman primates (NHPs) and humans. To better define the level/duration of chimerism required for stable renal allograft TOL, we retrospectively analyzed these parameters and compared them with transplant outcomes in NHP combined kidney and bone marrow transplant recipients. METHODS Peripheral blood levels and duration of myeloid or lymphoid chimerism were retrospectively analyzed in 34 NHP combined kidney and bone marrow transplantation recipients which were divided into 3 groups: TOL, n = 10; chronic antibody-mediated rejection (CAMR), n = 12; and T cell-mediated rejection (TCMR), n = 12. RESULTS All 4 of the recipients that failed to develop any chimerism lost their allografts due to TCMR after discontinuation of immunosuppression (56 ± 3 d). Among 30 recipients who successfully developed multilineage chimerism, 10 achieved long-term immunosuppression-free survival without rejection (1258 ± 388 d), 12 eventually developed CAMR (932 ± 155 d), and 8 developed TCMR (82 ± 10 d). The maximum level but not duration of lymphoid chimerism was significantly higher in TOL recipients compared with both CAMR (P = 0.0159) and TCMR (P = 0.0074). On the other hand, the maximum myeloid chimerism was significantly higher in TOL than in TCMR (P = 0.0469), but not in CAMR. Receiver operating characteristic analyses revealed that lymphoid chimerism levels of 3.1% or greater could reliably predict long-term immunosuppression-free renal allograft survival (P < 0.0001). CONCLUSIONS This retrospective study confirmed that induction of chimerism is essential for long-term immunosuppression-free survival, which best correlates with lymphoid chimerism levels higher than 3.1%.

Tolerance induction to prevent allograft rejection is a long-standing clinical goal. However, convincing and dependable tolerance identification remains elusive. Hypothesizing that intragraft RNA expression is informative in both rejection and tolerance, we profile intrarenal allograft RNA expression in a mixed chimerism renal allograft model of cynomolgus monkeys and identify biologically significant tolerance. Analysis of 67 genes identified 3 dominant factors, each with a different pattern of gene expressions, relating to T cell-mediated rejection (TCMR), chronic antibody-mediated rejection (CAMR), or Tolerance. Clustering these 3 factors created 9 groups. One of the 9 clustered groups, the Tolerance cluster, showed the lowest probability of terminal rejection, the longest duration of allograft survival, and the lowest relative risk of terminal rejection. The Tolerance factor consists of a novel set of gene expressions including cytokine and immunoregulatory genes adding mechanistic insights into tolerance. The Tolerance factor could not be identified within current pathologic diagnostic categories. The TCMR and CAMR factors are dominant to the Tolerance factor, causing rejection even if the Tolerance factor is present. These 3 factors determine the probability of terminal rejection or tolerance. This novel a posteriori approach permits identification of pathways of rejection, including tolerance.

RNA transcript expression estimates are a promising method to study the mechanisms and classification of renal allograft rejections. Here we use the Nanostring platform to profile RNA expression in renal allografts in a nonhuman primate (NHP), the Cynomolgus monkey. We analyzed protocol and indication 278 archival renal allograft samples, both protocol and indication from 76 animals with diagnoses of chronic antibody-mediated rejection (CAMR), acute cellular rejection (TCMR), and MIXED (both CAMR and TCMR), plus normals and samples with no pathological rejection using a Cynomolgus-specific probe set of 67 genes. Analysis identified RNA expression heterogeneity of endothelial and NK genes within CAMR and TCMR, including the stages of CAMR. Three factors were partitioned into additional groups. One group with the longest allograft survival time is pure CAMR without NK or CD3. Three mixed groups show variation in NK and CD3. TCMR was split into 2 groups with variation in NK genes. Additional validation of the complete gene-set correlated many of the genes with diagnoses of CAMR, MIXED, and TCMR rejections and with Banff histologic criteria defined in human subjects. These NHP data demonstrate the utility of RNA expression profiling to identify additional heterogeneity of endothelial and NK RNA gene expressions.

Tolerance of allografts achieved in mice via stable mixed hematopoietic chimerism relies essentially on continuous elimination of developing alloreactive T cells in the thymus (central deletion). Conversely, while only transient mixed chimerism is observed in nonhuman primates and patients, it is sufficient to ensure tolerance of kidney allografts. In this setting, it is likely that tolerance depends on peripheral regulatory mechanisms rather than thymic deletion. This implies that, in primates, upsetting the balance between inflammatory and regulatory alloimmunity could abolish tolerance and trigger the rejection of previously accepted renal allografts. In this study, six monkeys that were treated with a mixed chimerism protocol and had accepted a kidney allograft for periods of 1-10 years after withdrawal of immunosuppression received subcutaneous injections of IL-2 cytokine (0.6-3 × 10(6) IU/m(2) ). This resulted in rapid rejection of previously tolerated renal transplants and was associated with an expansion and reactivation of alloreactive pro-inflammatory memory T cells in the host's lymphoid organs and in the graft. This phenomenon was prevented by anti-CD8 antibody treatment. Finally, this process was reversible in that cessation of IL-2 administration aborted the rejection process and restored normal kidney graft function.

Biomarkers of transplant tolerance would enhance the safety and feasibility of clinical tolerance trials and potentially facilitate management of patients receiving immunosuppression. To this end, we examined blood from spontaneously tolerant renal transplant recipients and patients enrolled in two interventional tolerance trials using flow cytometry and gene expression profiling. Using a previously reported tolerant cohort as well as newly identified tolerant patients, we confirmed our previous finding that tolerance was associated with increased expression of B cell-associated genes relative to immunosuppressed patients. This was not accounted for merely by an increase in total B cell numbers, but was associated with the increased frequencies of transitional and naïve B cells. Moreover, serial measurements of gene expression demonstrated that this pattern persisted over several years, although patients receiving immunosuppression also displayed an increase in the two most dominant tolerance-related B cell genes, IGKV1D-13 and IGLL-1, over time. Importantly, patients rendered tolerant via induction of transient mixed chimerism, and those weaned to minimal immunosuppression, showed similar increases in IGKV1D-13 as did spontaneously tolerant individuals. Collectively, these findings support the notion that alterations in B cells may be a common theme for tolerant kidney transplant recipients, and that it is a useful monitoring tool in prospective trials.

While the induction of transient mixed chimerism has tolerized MHC-mismatched renal grafts in nonhuman primates and patients, this approach has not been successful for more immunogenic organs. Here, we describe a modified delayed-tolerance-induction protocol resulting in three out of four monkeys achieving long-term lung allograft survival without ongoing immunosuppression. Two of the tolerant monkeys displayed stable mixed lymphoid chimerism, and the other showed transient chimerism. Serial biopsies and post-mortem specimens from the tolerant monkeys revealed no signs of chronic rejection. The tolerant recipients also exhibited T cell unresponsiveness and a lack of alloantibody. This is the first report of durable mixed chimerism and successful tolerance induction of MHC-mismatched lungs in primates.

Despite advances in surgical technique and clinical care, lung transplantation still remains a short-term solution for the treatment of end-stage lung disease. To date, there has been limited experience in experimental lung transplantation using nonhuman primate models. Therefore, we have endeavored to develop a long-term, nonhuman primate model of orthotopic lung transplantation for the ultimate purpose of designing protocols to induce tolerance of lung grafts. Here, we report our initial results in developing this model and our observation that the nonhuman primate lung is particularly prone to rejection. This propensity toward rejection may be a consequence of 1) upregulated nonspecific inflammation, and 2) a larger number of pre-existing alloreactive memory T cells, leading to augmented deleterious immune responses. Our data show that triple-drug immunosuppression mimicking clinical practice is not sufficient to prevent acute rejection in nonhuman primate lung transplantation. The addition of horse-derived anti-thymocyte globulin and a monoclonal antibody to the IL-6 receptor allowed six out of six lung recipients to be free of rejection for over 120 days.

Mixed chimerism discovered in Freemartin cattle by Ray Owen 70 years ago paved the way for research on immune tolerance. Since his discovery, significant progress has been made in the effort to induce allograft tolerance via mixed chimerism in various murine models. However, induction of persistent mixed chimerism has proved to be extremely difficult in major histocompatibility complex mismatched humans. Chimerism induced in humans tends to either disappear or convert to full donor chimerism, depending on the intensity of the conditioning regimen. Nevertheless, our studies in both NHPs and humans have clearly demonstrated that renal allograft tolerance can be induced by transient mixed chimerism. Our studies have shown that solid organ allograft tolerance via transient mixed chimerism 1) requires induction of multilineage hematologic chimerism, 2) depends on peripheral regulatory mechanisms, rather than thymic deletion, for long-term maintenance, 3) is organ specific (kidney and lung but not heart allograft tolerance are feasible). A major advantage of tolerance induction via transient mixed chimerism is exclusion of the risk of graft-versus-host disease. Our ongoing studies are directed toward improving the consistency of tolerance induction, reducing the morbidity of the conditioning regimen, substituting clinically available agents, such as Belatacept for the now unavailable anti-CD2 monoclonal antibody, and extending the protocol to recipients of deceased donor allografts.

Since the first attempt of pig-to-primate liver xenotransplantation (LXT) in 1968, survival has been limited. We evaluated a model utilizing α-1,3-galactosyltransferase knockout donors, continuous posttransplant infusion of human prothrombin concentrate complex, and immunosuppression including anti-thymocyte globulin, FK-506, methylprednisone, and costimulation blockade (belatacept, n = 3 or anti-CD40 mAb, n = 1) to extend survival. Baboon 1 remained well until postoperative day (POD) 25, when euthanasia was required because of cholestasis and plantar ulcers. Baboon 2 was euthanized following a seizure on POD 5, despite normal liver function tests (LFTs) and no apparent pathology. Baboon 3 demonstrated initial stable liver function but was euthanized on POD 8 because of worsening LFTs. Pathology revealed C4d positivity, extensive hemorrhagic necrosis, and a focal cytomegalovirus inclusion. Baboon 4 was clinically well with stable LFTs until POD29, when euthanasia was again necessitated by plantar ulcerations and rising LFTs. Final pathology was C4d negative and without evidence of rejection, inflammation, or thrombotic microangiopathy. Thus, nearly 1-mo rejection-free survival has been achieved following LXT in two of four consecutive recipients, demonstrating that the porcine liver can support life in primates for several weeks and has encouraging potential for clinical application as a bridge to allotransplantation for patients with acute-on-chronic or fulminant hepatic failure.

Background Infusion of recipient regulatory T (Treg) cells promotes durable mixed hematopoietic chimerism and allograft tolerance in mice receiving allogeneic bone marrow transplant (BMT) with minimal conditioning. We applied this strategy in a Cynomolgus macaque model. Methods CD4 CD25 Treg cells that were polyclonally expanded in culture were highly suppressive in vitro and maintained high expression of FoxP3. Eight monkeys underwent nonmyeloablative conditioning and major histocompatibility complex mismatched BMT with or without Treg cell infusion. Renal transplantation (from the same BMT donor) was performed 4 months post-BMT without immunosuppression to assess for robust donor-specific tolerance. Results Transient mixed chimerism, without significant T cell chimerism, was achieved in the animals that received BMT without Treg cells (N = 3). In contrast, 2 of 5 recipients of Treg cell BMT that were evaluable displayed chimerism in all lineages, including T cells, for up to 335 days post-BMT. Importantly, in the animal that survived long-term, greater than 90% of donor T cells were CD45RA CD31, suggesting they were new thymic emigrants. In this animal, the delayed (to 4 months) donor kidney graft was accepted more than 294 days without immunosuppression, whereas non-Treg cell BMT recipients rejected delayed donor kidneys within 3 to 4 weeks. Early CMV reactivation and treatment was associated with early failure of chimerism, regardless of Treg cell administration. Conclusions Our studies provide proof-of-principle that, in the absence of early CMV reactivation (and BM-toxic antiviral therapy), cotransplantation of host Treg cell can promote prolonged and high levels of multilineage allogeneic chimerism and robust tolerance to the donor.

Type 1 primary hyperoxaluria (PH1) causes renal failure, for which isolated kidney transplantation (KT) is usually unsuccessful treatment due to early oxalate stone recurrence. Although hepatectomy and liver transplantation (LT) corrects PH1 enzymatic defect, simultaneous auxiliary partial liver transplantation (APLT) and KT have been suggested as an alternative approach. APLT advantages include preservation of the donor pool and retention of native liver function in the event of liver graft loss. However, APLT relative mass may be inadequate to correct the defect. We here report the first case of native portal vein embolization (PVE) to increase APLT to native liver mass ratio (APLT/NLM-R). Following initial combined APLT-KT, both allografts functioned well, but oxalate plasma levels did not normalize. We postulated the inadequate APLT/NLM-R could be corrected by trans-hepatic native PVE. The resulting increased APLT/NLM-R decreased serum oxalate to normal levels within 1 month following PVE. We conclude that persistently elevated oxalate levels after combined APLT-KT for PH1 treatment, results from inadequate relative functional capacity. This can be reversed by partial native PVE to decrease portal flow to the native liver. This approach might be applicable to other scenarios where partial grafts have been transplanted to replace native liver function.

Background Organ shortage is the greatest challenge facing the field of organ transplantation today. Use of more organs of marginal quality has been advocated to address the shortage. Method We examined the pattern of donation and organ use in the United States as shown in the Organ Procurement and Transplantation Network/United Network for Organ Sharing database of individuals who were consented for and progressed to organ donation between January 2001 and December 2010. Results There were 66,421 living donors and 73,359 deceased donors, including 67,583 (92.1 %) identified as donation after brain death and 5,776 (7.9 %) as donation after circulatory death (DCD).Comparing two periods, era 1 (01/2001-12/2005) and era 2 (01/2006-12/2010), the number of deceased donors increased by 20.3 %from33,300 to 40,059while there was a trend for decreasing living donation. The DCD subgroup increased from 4.9 to 11.7 % comparing the two eras. A significant increase in cardiovascular/cerebrovascular disease as a cause of deathwas also noted, from38.1 %in era 1 to 56.1 %in era 2 (p

We have previously reported that peri-transplant conditioning leads to successful induction of renal allograft tolerance via the mixed chimerism approach in nonhuman primates (NHP) and humans. However, this strategy requires treatments beginning six days prior to transplantation, which limits its relevance only to living donor transplant recipients. To extend the clinical applicability of this approach, we developed a novel regimen “delayed tolerance,” with which the recipient initially undergoes organ transplantation with conventional immunosuppression, followed by conditioning and donor bone marrow transplantation (DBMT) at a later date. This approach might be likened to “planting flowers in a battle field.” That is, the recipient’s immunologic environment after organ transplantation is like a battlefield filled with hostile innate and adaptive immune-responses directed against donor antigeneic specificities. Implanting fragile donor hematopoietic progenitors into this environment and encouraging them to bloom in this vicious field requires special treatments. In our NHP studies recently published in The American Journal of Transplantation, we showed that such “delayed tolerance,” in fact, can be induced in NHP through the mixed chimerism approach, if specific modifications to overcome/avoid donor-specific memory T cell responses are provided. These modifications include adequate depletion of CD8 memory T cells and timing of donor bone marrow administration to minimize levels of pro-inflammatory cytokines. This article addendum will provide a short summary of the original paper with our additional insights and interpretations.

There is a paucity of data on long-term outcomes of older kidney recipients. Our aim was to compare the early and long-term outcomes of deceased donor kidney transplantation in patients agedor=60 years with outcomes in younger recipients.From 1998 to 2005, we performed 271 deceased donor kidney transplants. There were 76 recipients (28.1%)60 years old. Older candidates were carefully selected based on their physiologic, cardiac, and performance status. Demographic data, including clinical characteristics, early complications, mortality, and patient and graft survival rates, were collected and analyzed.Older patients had comparable perioperative mortality and morbidity, incidence of delayed graft function (DGF), length of stay, and readmissions compared with younger patients. The rates of acute rejection and major infections were also comparable between the 2 study groups. Among older recipients, 25/76 (32.1%) patients received extended criteria donor kidneys compared with only 35/195 (17.9%) of younger patients (P.001). Nevertheless, equivalent 1-, 3-, and 5-year allograft survival rates were observed in elderly and young patients; 91.5% versus, 92.5%, 78.5% versus 81.9%, and 75.6% versus 78.5%, respectively. Overall patient survival was also comparable in both groups.Kidney transplantation in appropriately selected elderly recipients provides equivalent outcomes compared with those observed in younger patients. These observations support the notion that older recipients should not lose access to deceased donor kidney transplantation in the effort to achieve a perceived gain in social utility.

We have previously reported successful induction of transient mixed chimerism and long-term acceptance of renal allografts in MHC mismatched nonhuman primates. In this study, we attempted to extend this tolerance induction approach to islet allografts. A total of eight recipients underwent MHC mismatched combined islet and bone marrow (BM) transplantation after induction of diabetes by streptozotocin. Three recipients were treated after a nonmyeloablative conditioning regimen that included low-dose total body and thymic irradiation, horse Atgam (ATG), six doses of anti-CD154 monoclonal antibody (mAb), and a 1-month course of cyclosporine (CyA) (Islet A). In Islet B, anti-CD8 mAb was administered in place of CyA. In Islet C, two recipients were treated with Islet B, but without ATG. The results were compared with previously reported results of eight cynomolgus monkeys that received combined kidney and BM transplantation (Kidney A) following the same conditioning regimen used in Islet A. The majority of kidney/BM recipients achieved long-term renal allograft survival after induction of transient chimerism. However, prolonged islet survival was not achieved in similarly conditioned islet/BM recipients (Islet A), despite induction of comparable levels of chimerism. In order to rule out islet allograft loss due to CyA toxicity, three recipients were treated with anti-CD8 mAb in place of CyA. Although these recipients developed significantly superior mixed chimerism and more prolonged islet allograft survival (61, 103, and 113 days), islet function was lost soon after the disappearance of chimerism. In Islet C recipients, neither prolonged chimerism nor islet survival was observed (30 and 40 days). Significant improvement of mixed chimerism induction and islet allograft survival were achieved with a CyA-free regimen that included anti-CD8 mAb. However, unlike the kidney allograft, islet allograft tolerance was not induced with transient chimerism. Induction of more durable mixed chimerism may be necessary for induction of islet allograft tolerance.

We sought to determine the effects of exogenous administration of human coagulation factors following pig-to-baboon liver xenotransplantation (LXT) using GalT-KO swine donors. After LXT, baboons received no coagulation factors (historical control, n = 1), bolus administration of a human prothrombin concentrate complex (hPCC; 2.5 mL/kg, n = 2), continuous infusion of hPCC (1.0 mL/h, n = 1) or continuous infusion of human recombinant factor VIIa (1 µg/kg per hour, n = 3). The historical control recipient demonstrated persistent thrombocytopenia despite platelet administration after transplant, along with widespread thrombotic microangiopathy (TMA). In contrast, platelet levels were maintained in bolus hPCC recipients; however, these animals quickly developed large-vessel thrombosis and TMA, leading to graft failure with shortened survival. Recipients of continuous coagulation factor administration experienced either stabilization or an increase in their circulating platelets with escalating doses. Furthermore, transfusion requirements were decreased, and hepatic TMA was noticeably absent in recipients of continuous coagulation factor infusions compared with the historical control and bolus hPCC recipients. This effect was most profound with a continuous, escalating dose of factor VIIa. Further studies are warranted because this regimen may allow for prolonged survival following LXT.

Six patients with renal failure due to multiple myeloma (MM) received simultaneous kidney and bone marrow transplantation (BMT) from HLA-identical sibling donors following nonmyeloablative conditioning, including cyclophosphamide (CP), peritransplant antithymocyte globulin and thymic irradiation. Cyclosporine (CyA) was given for approximately 2 months posttransplant, followed by donor leukocyte infusions. All six patients accepted their kidney grafts long-term. Three patients lost detectable chimerism but accepted their kidney grafts off immunosuppression for 1.3 to >7 years. One such patient had strong antidonor cytotoxic T lymphocyte (CTL) responses in association with marrow rejection. Two patients achieved full donor chimerism, but resumed immunosuppression to treat graft-versus-host disease. Only one patient experienced rejection following CyA withdrawal. He responded to immunosuppression, which was later successfully withdrawn. The rejection episode was associated with antidonor Th reactivity. Patients showed CTL unresponsiveness to cultured donor renal tubular epithelial cells. Initially recovering T cells were memory cells and were enriched for CD4+CD25+ cells. Three patients are in sustained complete remissions of MM, despite loss of chimerism in two. Combined kidney/BMT with nonmyeloablative conditioning can achieve renal allograft tolerance and excellent myeloma responses, even in the presence of donor marrow rejection and antidonor alloresponses in vitro.

Objective—To obtain information on menstrual patterns before and after transplantation, desire for future pregnancy, and use of contraception among premenopausal kidney transplant recipients.Study Design—This observational study collected information using self-administered anonymous questionnaires during a routine outpatient clinic visit.Results—Of the 107 women who completed the questionnaire, 41 identified themselves as being premenopausal. Among the 41 premenopausal women, approximately half of the women reported their current menstrual patterns as normal and 26% were not using any form of contraception. Overall, 10 women (24%) reported a desire to become pregnant and 4 women (10%) had a successful pregnancy after transplantation. Most of the women who desired a future pregnancy (8/10) were receiving an immunosuppressive regimen that included mycophenolate mofetil.Conclusion—Kidney transplantation in the current era is associated with a return of normal menstrual function in the majority of female tra...

Two patients with end-stage renal disease secondary to multiple myelomas were treated with combined kidney and bone marrow transplantation in an effort to achieve donor-specific allotolerance through the induction of mixed lymphohematopoietic chimerism.

Cadaver sources are insufficient for the increasing demand for liver transplantation. Right-lobe liver transplantation from living donors is fully developed in Japan and has been rapidly increasing in the United States during the past 2 years, although donor risk is greater than in other types of solid organ transplantation. The authors examine the psychiatric and ethical aspects of right-lobe liver transplantation in light of cultural differences between the United States and Japan.

Sickle cell intrahepatic cholestasis is a potentially fatal end-organ complication of sickle cell anemia. Renal involvement in sickle cell anemia is common, and in some cases, can present as acute renal failure. Although renal transplants have been performed in patients with sickle cell anemia since the late 1960s and a number of liver transplants have been recently performed for these complications, there has not been experience with dual organ transplantation for sickle cell anemia-related complications. We describe the case of a patient with sickle cell anemia who underwent successful combined liver and kidney transplantation after the development of acute sickle cell intrahepatic cholestasis and renal failure requiring continuous venovenous hemodialysis. The patient underwent a successful combined liver and kidney transplant with limited perioperative complications and preserved allograft function. At 22 months posttransplant, the patient expired as a result of an acute pulmonary embolus in the setting of bilateral hip fractures. Autopsy revealed no evidence of liver or kidney allograft rejection and evidence of chronic sickle cell nephropathy in the native kidney. Combined liver and kidney transplantation is a viable therapeutic option in patients with severe end-organ effects of sickle cell anemia.

We have previously reported successful induction of renal allograft tolerance via a mixed chimerism approach in nonhuman primates. In those studies, we found that costimulatory blockade with anti-CD154 mAb was an effective adjunctive therapy for induction of renal allograft tolerance. However, since anti-CD154 mAb is not clinically available, we have evaluated CTLA4Ig as an alternative agent for effecting costimulation blockade in this treatment protocol. Two CTLA4Igs, abatacept and belatacept, were substituted for anti-CD154 mAb in the conditioning regimen (low dose total body irradiation, thymic irradiation, anti-thymocyte globulin and a 1-month posttransplant course of cyclosporine [CyA]). Three recipients treated with the abatacept regimen failed to develop comparable lymphoid chimerism to that achieved with anti-CD154 mAb treatment and these recipients rejected their kidney allografts early. With the belatacept regimen, four of five recipients developed chimerism and three of these achieved long-term renal allograft survival (>861, >796 and >378 days) without maintenance immunosuppression. Neither chimerism nor long-term allograft survival were achieved in two recipients treated with the belatacept regimen but with a lower, subtherapeutic dose of CyA. This study indicates that CD28/B7 blockade with belatacept can provide a clinically applicable alternative to anti-CD154 mAb for promoting chimerism and renal allograft tolerance.

Background. Pseudomonas aeruginosa is an uncommon cause of infection in the female genital tract. We report a case of postmenopausal tubo-ovarian abscess (TOA) due to P. aeruginosa in a renal transplant recipient. The presentation included mild abdominal symptoms with rapid progression of peritonitis and surgical abscess drainage. This is the first such case in an organ transplant recipient described in the English literature. Methods and Results. Published reports of 1040 cases of TOA were reviewed. The most common features were a history of sexually transmitted disease or pelvic inflammatory disease, and symptoms including abdominal pain and fever. Escherichia coli, Bacteroides spp., and Klebsiella pneumoniae were the most frequently encountered pathogens. Neisseria gonorrhoeae and Chlamydia trachomatis, which are frequently isolated from cervical cultures, are uncommonly isolated from tubo-ovarian abscesses. Forty percent of patients were treated with antibiotics alone, 18.8% with abdominal surgery, and 32% with surgery and antimicrobial therapy. Conclusion. This report illustrates the muted presentation and atypical microbiology of gynecologic infection in an organ transplant recipient.

Background. Recent studies suggest an association between diabetes mellitus and hepatitis C virus (HCV) infection. Our aim was to determine (1) the prevalence and determinants of new onset posttransplant diabetes mellitus (PTDM) in HCV (+) liver transplant (OLT) recipients, (2) the temporal relationship between recurrent allograft hepatitis and the onset of PTDM, and (3) the effects of antiviral therapy on glycemic control. Methods. Between January of 1991 and December of 1998, of 185 OLTs performed in 176 adult patients, 47 HCV (+) cases and 111 HCV (-) controls were analyzed. We reviewed and analyzed the demographics, etiology of liver failure, pretransplant alcohol abuse, prevalence of diabetes mellitus, and clinical characteristics of both groups. In HCV (+) patients, the development of recurrent allograft hepatitis and its therapy were also studied in detail. Results. The prevalence of pretransplant diabetes was similar in the two groups, whereas the prevalence of PTDM was significantly higher in HCV (+) than in HCV (-) patients (64% vs. 28%, P=0.0001). By multivariate analysis, HCV infection (hazard ratio 2.5, P=0.001) and methylprednisolone boluses (hazard ratio 1.09 per bolus, P=0.02) were found to be independent risk factors for the development of PTDM. Development of PTDM was found to be an independent risk factor for mortality (hazard ratio 3.67, P

UNLABELLED Development of mixed chimerism by donor bone marrow transplantation (DBMT) has led to long-term tolerance of solid organ allografts in nonhuman primates. As an initial attempt to extend this approach to cellular transplant, islet transplant from the same donor was attempted in the recipient previously made tolerant to a kidney allograft. METHODS After the conditioning with ATG, total body irradiation, thymic irradiation, and splenectomy, DBMT was performed followed by 4 weeks of cyclosporine. Kidney transplantation and native nephrectomies were subsequently performed on day 89. After 2.8 years of DBMT, diabetes was induced by streptozocin (STZ) and islets from bone marrow and kidney donor were transplanted without immunosuppression. RESULTS After DBMT, the recipient developed chimerism and no evidence of kidney rejection for more than 1000 days. STZ induced diabetes was reversed after the islet transplantation. Islet biopsies demonstrated insulin staining without rejection. Although the recipient became diabetic 300 days after islet transplantation, viable transplanted islets were found in the liver and under the kidney capsule without any evidence of rejection. CONCLUSION Tolerance with a nonmyeloablative conditioning can allow successful pancreatic islet transplantation without immunosuppression. Because no histological evidence of rejection was identified, recurrent diabetes is presumed to be inadequate islet mass.

Background. Complement activation has recently been implicated as a contributing factor to early and late allograft dysfunction in cardiac transplantation. The current study was designed to determine whether measurement of plasma complement fragments C4d and SC5b-9 would be useful in detecting acute rejection or accelerated graft atherosclerosis (AGA) in cardiac allograft recipients. Methods. We measured complement activation products, C4d (classical pathway) and SC5b-9 (terminal pathway), at the time of routine endomyocardial biopsy in heart transplant recipients. Ten patients in the immediate posttransplantation period (0-100 days) and 19 patients more than 6 months after transplantation were studied. Results. No correlation was found between plasma levels of complement activation fragments and the presence of biopsy-proven acute allograft rejection or AGA (assessed by coronary angiography). However, plasma C4d and SC5b-9 were significantly elevated in 9 of 10 and 7 of 10 patients, respectively, in the immediate posttransplantation period. This was followed by progressive decrease in the levels of C4d and SC5b-9 fragments during the first 4-6 weeks after transplantation. Conclusion. We conclude that measuring plasma levels of fragments C4d and SC5b-9 is not a useful noninvasive method for detecting acute rejection or AGA after heart transplantation. However, this study provides further evidence that early complement activation after heart transplantation may play a pathogenic role in allograft injury.

Background. Nonmyeloablative T cell depletion followed by donor bone marrow infusion has proved to be an effective approach to induction of mixed chimerism and tolerance of organ allografts in non-human primates. To help define the mechanisms involved we have compared T cell depletion with ATG versus anti-CD2 monoclonal antibody with respect to establishment of mixed chimerism and induction of tolerance. Background. Method. Background. Both nonmyeloablative regimens included low dose total body irradiation (1.5 Gy x 2), thymic irradiation (7 Gy), splenectomy and kidney plus donor bone marrow transplantation, followed by a 4-week posttransplant course of cyclosporine. In addition, the ATG group (13 recipients) received antithymocyte globulin, although the LOCD2b group (10 recipients) were treated with an anti-CD2 monoclonal antibody (LOCD2b). Results. In the ATG group, 11 of 13 monkeys developed multilineage chimerism and 9 survived for more than 100 days without kidney allograft rejection. In contrast, 0/10 monkeys in the LOCD2b group developed chimerism, 5 died of infection and 5 suffered progressive rejection; only 1 recipient survived beyond 100 days. Sequential monitoring of peripheral blood mononuclear cells revealed greater T cell (CD3+) depletion in the LOCD2b-treated animals compared to those receiving ATG. However, NK cells (CD16+CD8+) were significantly more depleted in the ATG group and NK function remained abrogated longer after ATG than LOCD2b treatment (3 weeks vs. Conclusion. Despite excellent T cell depletion by LoCD2b, ATG was more effective in inducing chimerism and tolerance. This difference correlated with anti-NK activity of the two reagents. These data suggest that NK cells may also resist engraftment of allogeneic bone marrow cells in this model.

Background: Although the primary treatment of symptomatic cytomegalovirus (CMV) disease in organ transplant recipients is successful in >90% of individuals, relapsing disease, particularly in those with primary infection, remains an important problem. Previously, we had observed that the rate of symptomatic recurrence was >60% in those with primary disease (seronegative for CMV prior to transplant), and approximately 20% in those who were seropositive prior to transplant. The present study was undertaken to determine whether a maintenance regimen of oral ganciclovir for 2–3 months added to the routine 14–21 days of intravenous ganciclovir would further prevent symptomatic CMV recurrence. Methods: From May 1995 until June 1998, all kidney and liver transplant recipients with confirmed tissue-invasive CMV disease or CMV syndrome were treated with 14–21 days of intravenous ganciclovir (5 mg/kg b.i.d. with dose adjusted for renal dysfunction) followed by 2–3 months of oral ganciclovir (2 g daily). The incidence of recurrence of CMV disease and/or viremia during and after oral therapy was then determined over a mean follow-up of 530.6 days. Results: Thirty-seven patients, 19 kidney and 18 liver transplant recipients, were studied; 5 had biopsy-proven tissue-invasive disease (13.5) and 32 suffered a CMV syndrome (86.5). Twenty-one of these patients (58.6) were seronegative for CMV prior to transplant and received an allograft from a seropositive donor (D+/R−). Overall, 10 patients (27.0) developed CMV recurrence. Eight of 21 patients who were D+/R− for CMV (38.1) developed recurrence as opposed to 2 of 16 patients with other serologic status (12.5) (P=0.14). Patients with recurrent CMV disease and/or viremia had a peak antigenemia assay titer during their initial CMV event of 319.2 positive cells/2 slides compared with 109.8 positive cells/2 slides for patients without recurrent CMV infection (P=0.14); the trend of having a higher peak antigenemia assay titer among patients who recurred occurred both in patients who were at risk of primary CMV infection (D+/R− for CMV) and in those who were not. Two patients developed recurrent infection with strains of CMV that were resistant to ganciclovir. Conclusions: This new therapeutic regimen of oral ganciclovir following intravenous ganciclovir slightly reduced the overall rate of recurrent CMV disease and/or viremia, but it still did not adequately prevent CMV recurrence in patients who are at risk of primary infection prior to transplant. Of particular concern, 2 patients with primary infection treated with this regimen developed ganciclovir-resistant recurrent disease ( Note).

BACKGROUND Leukopenia is not infrequently encountered following solid organ transplantation, most often in the setting of cytomegalovirus (CMV) disease and/or its treatment with ganciclovir. The present study was undertaken to determine the safety and efficacy of granulocyte colony-stimulating factor (G-CSF) in renal and liver transplant recipients with leukopenia. METHODS Between 1 June 1991 and 1 June 1998, patients received G-CSF for 2 indications: 1) white blood cell count (WBC) < 3000/mm3, with a decline from baseline; 2) to shorten the duration of leukopenia associated with chemotherapy. A retrospective review of the outcome of such therapy was undertaken. RESULTS 50 patients were given 100 courses of treatment with G-CSF; 35 of 168 liver transplant recipients (20.8%), 14 of 391 kidney transplant recipients (3.6%), and 1 of 4 recipients of combined liver-kidney transplants (25.0%) received from 1 to 9 courses of G-CSF. Presumed causes of leukopenia were identified as ganciclovir in 28 cases (28.0%), CMV in 21 (21.0%), chemotherapy in 12 (12.0%), sepsis in 11 (11.0%), azathioprine in 5 (5.0%), interferon in 3 (3.0%) and other causes in 20 cases (20.0%). The median length of therapy was 10.0 days (range 1-154 days) and the average dose of daily G-CSF received was 3.9+/-1.5 microg/kg/day. The average WBC was (2.4+/-1.3 )x 10(3)/microl at the beginning of therapy, and (13.8+/-9.1) x 10(3)/microl at the end of therapy. In 7 of 100 treatments (7.0%) a WBC of 5.0 x 10(3)/microl was not reached during G-CSF therapy; in 6 of these 7 cases, G-CSF therapy lasted fewer than 4 days. The mean time needed to reach a WBC count of 5 x 10(3)/microl was 3.7+/-3.3 days among 71 patients who had daily WBC counts sent. Eight G-CSF treatments (8.0%) were followed by episodes of rejection appearing during or within 2 months of treatment; 5 of them were biopsy-documented. No relation was found between the highest WBC obtained during G-CSF therapy and the risk of rejection. Eight patients (16.0%) died while receiving G-CSF, all from infection. Six of these 8 patients were receiving G-CSF for leukopenia secondary to sepsis. Overall, 25 patients (50.0%) received 49 courses of G-CSF secondary to CMV and/or ganciclovir therapy. In 40 of 49 courses (81.6%), ganciclovir could be continued at recommended doses. Twenty-one of 22 patients (95.5%) with symptomatic CMV infection had a clinical response to ganciclovir. Sixteen of 18 patients (88.9%) treated for a CMV infection and followed with serial antigenemia assays attained microbiological cure; both patients who did not were infected with ganciclovir resistant CMV. CONCLUSION G-CSF was well tolerated in solid organ transplant recipients. It was particularly useful in patients with CMV disease, allowing optimal ganciclovir therapy.

Background. The optimal regimen for the prevention and treatment of cytomegalovirus (CMV) disease in solid organ transplant recipients remains to be defined, particularly for patients with abnormal or changing renal function. Methods. A prospective trial was conducted in patients receiving i.v. ganciclovir using a standardized dosing nomogram that corrects for renal function. Steady state peak (P) and trough (T) serum levels were determined by high-performance liquid chromatography and correlated with therapeutic outcomes and toxicities attributable to ganciclovir. Results. Over the study period, 44 individuals received ganciclovir prophylaxis (5 mg/kg/day) and 25 patients were treated (5 mg/kd q12 hr) for symptomatic CMV disease. Ganciclovir levels (mg/ml6SD) achieved in prophylaxis were P: 7.9863.34, T: 3.0362.63; and in treatment were P: 9.0063.72, T: 2.6561.82. Despite corrections for renal dysfunction, undialyzed patients with serum creatinine >3.0 mg/dl had trough levels in excess of the population mean (T: range 3‐ 8 mg/ml). Failure of prophylaxis (disease) or therapy (relapse) occurred in 14 patients; 8 of these were at risk for primary infection (donor CMV seropositive, recipient seronegative, P 3.0, 1.25 mg/kg QOD for Cr >5.0). Some groups of transplant recipients may require more intensive anti-CMV regimens. Cytomegalovirus (CMV*) is a major cause of morbidity in solid organ transplant recipients (1‐9). In various series, between 20 and 70% of organ transplant recipients are affected by CMV infection (2, 10 ‐12). Ganciclovir, in both i.v. and oral formulations, is commonly used to prevent and to treat CMV infection, although the optimal dose and duration of therapy have not yet been completely defined (13‐21). In clinical practice, patients requiring prophylaxis or treatment for CMV infection often have abnormal or fluctuating renal function. This is particularly relevant in the setting of recent organ transplantation or during acute renal allograft rejection. However, few data are available on which to base decisions about the dosing of ganciclovir in such patients (22‐23). In the attempt to optimize antiviral therapy in solid organ transplant recipients with variable renal function, a prospective study was performed to study blood levels and clinical outcomes associated with the routine administration of i.v. ganciclovir based on a standardized nomogram. Peak and trough serum ganciclovir levels were measured by highpressure liquid chromatography. An attempt was made to assess the dosing of ganciclovir and to assess the impact of dosage and serum levels on the efficacy and toxicities of antiviral therapy and any impact that might be detected with the use of prophylactic or therapeutic ganciclovir on the development of or recurrence of symptomatic CMV infection.

Background. We have previously demonstrated that induction of mixed lymphohematopoietic chimerism resulted in donor specific renal allograft tolerance without the need for chronic immunosuppression in nonhuman primates. Here we have tested whether tolerance can be similarly induced for baboon to cynomolgus renal xenografts. Methods. After preconditioning with anti-thymocyte globulin (ATG), nonlethal total body irradiation, and thymic irradiation, cynomolgus monkeys underwent splenectomy, native nephrectomies, and baboon marrow and renal transplants. Postoperative cyclosporine was given for 28 days. Results. In Group 1 (n=2, survival=13, 14 days), both animals developed anti-donor immunoglobulin G, had biopsy findings consistent with humoral rejection, and showed rapidly progressive xenograft failure. In Group 2 (n=5, survival=1, 16, 33, 112, 190 days), 15-deoxyspergualine was added to the regimen (Day 0-13). In one long-term survivor, donor specific hyporesponsiveness was first observed (mixed lymphocyte culture [(MLR]) on Day 48. MLR reactivity returned on Day 64 together with the development of anti-donor antibody and subsequent xenograft failure on Day 112. Donor specific T-cell hyporesponsiveness was detected in the other long-term survivor for the first 133 days, after which a donor-specific skin xenograft was placed, (survival 24 days). Following the skin graft rejection, a rise in the MLR, development of anti-donor antibody and progressive rejection of the renal xenograft were observed. Conclusions. Antibody-mediated rejection seems to constitute the major difference between concordant xenografts and allografts. Addition of 15-deoxyspergualine for 2 weeks posttransplant extended concordant primate xenograft survival to 6 months without chronic immunosuppression. In contrast to the allogeneic model, renal transplant acceptance in this xenogeneic system was interrupted by placement of a donor-specific skin graft.

Background Multilineage chimerism and long-term acceptance of renal allografts has been produced in non-human primates conditioned with a nonmyeloablative regimen. Our study was undertaken to evaluate the immunological and pathological status of long-term survivors and to define the role of splenectomy and of the primarily vascularized kidney in the regimen. Method Monkeys were treated with the basic regimen, including: total body irradiation, thymic irradiation, antithymocyte globulin, donor bone marrow transplantation, and a 4-week course of cyclosporine after which no further immunosuppression was given. They were divided into four groups according to the timing of kidney transplantation (KTx) and splenectomy as follows; group A (n=13): KTx and splenectomy on the day of donor bone marrow transplantation (day 0); group B (n=3): KTx on day 0 without splenectomy; group C (n=7): splenectomy on day 0 but delayed KTx until 3 to 16 weeks post-donor bone marrow transplantation; group D (n=3): both splenectomy and KTx delayed until day 120 post-donor bone marrow transplantation. Results In group A, 11 of 13 monkeys developed chimerism and 9 monkeys achieved long-term survival of 4 to 70 months without evidence of chronic vascular rejection. Alloantibodies were detected in only one long-term survivor. In contrast, all three monkeys in group B developed alloantibodies and rejected their allografts. In group C, long-term survival without alloantibody production was observed in two of three monkeys that had developed chimerism. In group D, all three recipients were sensitized and rejected the kidney allografts rapidly after transplantation. Conclusions 1) Production of anti-donor antibody was prevented in most recipients that developed mixed chimerism in the regimens with splenectomy at the time of donor bone marrow transplantation. 2) If splenectomy is not included in the initial conditioning regimen, induction of B cell tolerance is less likely and the result is late onset of alloantibody production and allograft rejection. 3) Immediate transplantation of the kidney at the time of recipient conditioning is not essential for induction of donor specific hyporesponsiveness by bone marrow transplantation.

The distinction between acute humoral rejection (AHR) and acute cellular rejection (ACR) in renal allografts is therapeutically important, but pathologically difficult. Since AHR is probably mediated by antibodies to the donor endothelium that activate the classical complement pathway, it was hypothesized that peritubular capillary C4d deposition might distinguish this group. Renal biopsies (n = 16) from 10 patients with AHR who had acute graft dysfunction, neutrophils in peritubular capillaries, and a concurrent positive cross-match were stained for C4d by immunofluorescence. Control biopsies for comparison showed ACR (n = 14), cyclosporin A toxicity (n = 6), or no abnormality (n = 4). Peribiopsy sera were tested for anti-donor HLA antibody. C4d deposited prominently and diffusely in the peritubular capillaries in all AHR biopsies (16 of 16). IgM and/or C3 were also present in 19 and 44%, respectively. With two-color immunofluorescence, C4d was localized in basement membranes (type IV collagen+) and in the endothelium (Ulex europaeus agglutinin-I+). In ACR, no more than trace C4d was found in peritubular capillaries (P < 0.0001 versus AHR), and no patient had anti-donor HLA antibodies (0 of 8); 27% had neutrophils in peritubular capillaries. One of six biopsies with cyclosporin A toxicity had similar C4d deposits, and circulating anti-donor class I antibody was detected. Grafts with AHR were lost (40%) more often than those with ACR (0%; P < 0.02). C4d in peritubular capillary walls distinguishes AHR from ACR, is more specific and sensitive than traditional criteria, and is a potentially valuable adjunct in the diagnosis of graft dysfunction.

BACKGROUND: Mixed allogeneic hematopoietic chimerism has previously been reliably achieved and shown to induce tolerance to fully MHC-mismatched allografts in mice and monkeys. However, the establishment of hematopoietic chimerism has been difficult to achieve in the discordant pig-to-primate xenogeneic model. METHODS: To address this issue, two cynomolgus monkeys were conditioned by whole body irradiation (total dose 300 cGy) 6 and 5 days before the infusion of pig bone marrow (BM). Monkey anti-pig natural antibodies were immunoadsorbed by extracorporeal perfusion of monkey blood through a pig liver, immediately before the intravenous infusion of porcine BM (day 0). Cyclosporine was administered for 4 weeks and 15-deoxyspergualin for 2 weeks. One monkey received recombinant pig cytokines (stem cell factor and interleukin 3) for 2 weeks, whereas the other received only saline as a control. RESULTS: Both monkeys recovered from pancytopenia within 4 weeks of whole body irradiation. Anti-pig IgM and IgG antibodies were successfully depleted by the liver perfusion but returned to pretreatment levels within 12-14 days. Methylcellulose colony assays at days 180 and 300 revealed that about 2% of the myeloid progenitors in the BM of the cytokine-treated recipient were of pig origin, whereas no chimerism was detected in the BM of the untreated control monkey at similar times. The chimeric animal was less responsive by mixed lymphocyte reaction to pig-specific stimulators than the control monkey and significantly hyporesponsive when compared with a monkey that had rejected a porcine kidney transplant. CONCLUSION: To our knowledge, this is the first report of long-term survival of discordant xenogeneic BM in a primate recipient.

Background. Due to the limited supply and increased demand for donor livers, waiting times are progressively lengthening, which may lead to transplantation at more advanced and less cost-effective stages of disease. The purpose of this study was to evaluate the outcomes and hospital charges of liver transplantation during two recent eras to identify areas for providing more cost-effective care. Methods. A total of 144 primary liver allografts were performed from 1991 to 1996. Patient characteristics, outcome measures, and hospital charges were compared for patients receiving allografts between 1991 and 1993 (group A) versus those receiving grafts between 1994 and 1996 (group B) using unpaired Student t tests for continuous data and chi-squared tests for categorical data. Results. In comparing groups A and B, no significant differences in patient demographics, relative contraindications, or indication for transplantation existed; median waiting time from date of listing until transplant increased from 88 days to 159 days; and a shift in UNOS priority status at time of transplantation occurred, as the percentage of patients requiring inpatient care increased from 58% to 75% (P=0.034). Despite this, patient hospital and 1-year survival significantly improved from 75.0% to 90.3% (P=0.016), and from 68.1% to 88.9% (P=0.002), respectively. Total hospital charges, without correction for inflation, were $174,908

Background Xenotransplantation would provide a solution to the current shortage of organs for transplantation. Our group has been successful in inducing tolerance in mice and monkey models of allogeneic transplantation. The present study attempts to extend the same tolerance-inducing regimen to a pig-to-baboon organ transplantation model. Methods Nine baboons underwent a conditioning regimen (consisting of nonmyeloablative or myeloablative whole body and thymic irradiation, splenectomy, antithymocyte globulin, pharmacologic immunosuppression and porcine bone marrow transplantation [BMTx]), which has previously been demonstrated to induce donor-specific allograft tolerance in monkeys. In addition, immunoadsorption of anti-alphaGal antibody (Ab) was performed. Four of the nine baboons received pig kidney transplants (KTx), and one also underwent repeat transplantation with an SLA-matched kidney. Two received heterotopic pig heart transplants (HTx). Three baboons underwent conditioning without organ transplantation for long-term studies of natural Ab kinetics. Results In the three baboons that received the conditioning regimen without an organ transplant, immunoadsorption reduced Ab by approximately 90%, but recovery of Ab to pretreatment level or higher occurred within 7 days. In contrast, the level of Ab remained low after organ transplant. No Ab to pig antigens other than alphaGal was detected in any baboon before or after BMTx, KTx, or HTx. No graft succumbed to hyperacute rejection. KTx function began to deteriorate within 3-6 days, with oliguria and hematuria progressing to anuria, and the kidneys were excised after 3, 6, 9, 11, and 14 days, respectively. One HTx ceased functioning at 8 days; the second baboon died with a contracting HTx at 15 days. Features of coagulopathy and thrombocytopenia developed in all six transplanted baboons (high D-dimer, prolonged prothrombin time and partial thromboplastin time, and falling fibrinogen) resulting in serious bleeding complications in two baboons, one of which died on day 9. Donor organs showed progressive acute humoral rejection with deposits of IgM, IgG, and complement; a focal mononuclear cellular infiltrate was also observed. The ureter was the earliest structure of the KTx affected by rejection, with progression to necrosis. Conclusions This conditioning regimen prevented hyperacute rejection but was ineffective in preventing the return of Ab, which was associated with the development of acute humoral rejection with features of coagulopathy. No baboon developed anti-pig Ab other than alphaGal Ab. Further modifications of the protocol directed toward suppression of production of Ab are required to successfully induce tolerance to pig organs in baboons.

Background. OKT3 monoclonal antibody therapy results in an acute clinical syndrome (ACS) associated with the release of tumor necrosis factor and sequestration of neutrophils in the lungs. We have previously shown that inhibition of tumor necrosis factor does not completely eliminate OKT3-ACS, suggesting that other factors also contribute to the ACS. The current studies analyzed complement activation in vivo during the first hour after OKT3 administration. Methods. Renal (n=4) and lung (n=4) transplant recipients received OKT3 as treatment for rejection and induction therapy, respectively. Complement activation products C4d, Bb, iC3b, and SC5b-9 were measured by ELISA. Hemodynamic parameters were also monitored in the lung transplant recipients. Neutrophil expression of CD11a, CD11b, and CD18 was monitored by flow cytometry. Controls included patients receiving methylprednisolone for rejection (n=4), two adults with adult respiratory distress syndrome who received extracorporeal membrane oxygenation, and normal volunteers (n=5). P values less than 0.05 ( * ) were considered significant. Results. Increases in the plasma levels of C4d, Bb, iC3b, and SC5b-9 were observed in seven of eight patients after OKT3 administration. Mean values (n=8) at 0, 15, and 60 min (in μg/ml) were as follows-C4d: 1.865, 2.644 * , and 2.607 * ; Bb: 0.245, 0.411, and 0.385; iC3b: 10.881, 17.242 * , and 15.145 * ; and SC5b-9: 0.232, 0.269, and 0.302 * . An increase in CD11b and CD18 and a decrease of CD11a on neutrophils in parallel with complement activation was observed. In lung transplant recipients, C3 activation correlated with increases in mean pulmonary and central venous pressures (P

BACKGROUND Intravascular fibrin deposition and platelet sequestration occur with porcine xenograft rejection by baboons. Disseminated intravascular coagulopathy may arise either as a direct consequence of the failure to fully deplete xenoreactive natural antibodies and block complement, or because of putative cross-species molecular incompatibilities in this discordant species combination. METHODS Three baboons were conditioned with retrovirally transduced autologous bone marrow to induce tolerance to swine antigens. Xenoreactive natural antibodies and complement were depleted by plasmapheresis and the use of Gal alpha1-3Gal column adsorptions; baboons were then splenectomized and underwent renal xenografting from inbred, miniature pigs. Soluble complement receptor type-1 with protocol immunosuppression (mycophenolate mofetil, 15-deoxyspergualin, steroids, and cyclosporine) was administered. RESULTS A bleeding diathesis was clinically evident from days 5 to 12 after transplantation in two baboons. Low levels of circulating C3a, C3d, and iC3b were measured despite the absence of functional circulating complement components. Profound thrombocytopenia with abnormalities in keeping with disseminated intravascular coagulopathy were observed. Prolongation of prothrombin and partial thromboplastin times was accompanied by evidence for tissue factor-mediated coagulation pathways, high levels of thrombin generation (prothrombin fragment F(1+2) production and thrombin-antithrombin complex formation), fibrinogen depletion, and production of high levels of the fibrin degradation product D-dimer. Importantly, these disturbances resolved rapidly after the excision of the rejected xenografts in two surviving animals. Histopathological examination of the rejected xenografts confirmed vascular injury, fibrin deposition, platelet deposition, and localized complement activation. CONCLUSIONS Systemic coagulation disturbances are associated with delayed xenograft rejection.

Background, Organ transplant recipients who are seropositive for cytomegalovirus (CMV) and who are treated with antilymphocyte antibody (ALA) therapy have a high rate of symptomatic CMV disease. The intravenous administration of ganciclovir therapy once daily during ALA therapy decreased the incidence from 24% to 10% in patients receiving ALA as an induction therapy and from 64% to 22% in those treated for rejection. The present study was undertaken to determine whether a more intensive and sustained antiviral regimen could be more effective. Methods. From April 1995 to December 1997, all CMV seropositive renal and liver transplant recipients who received ALA therapy were treated with intravenously administered ganciclovir (5 mg/kg/day with dose adjusted for renal dysfunction) for the length of ALA therapy and then with orally administered acyclovir (400 mg three times/day) or ganciclovir (1 gm twice/day) for 3 to 4 months. The incidence of CMV viremia and of CMV disease was determined during the 6 months after completion of ALA therapy. Results. Forty-one patients (35 renal and 6 liver transplant recipients) were studied. CMV disease occurred in 2 patients (4.9%), both of whom were treated for rejection; it occurred in 1 of 21 patients (4.8%) treated with orally administered acyclovir, and in 1 of 20 patients (5%) treated with orally administered ganciclovir. The only patient who developed CMV disease in the ganciclovir group had received only 26 days of oral antiviral therapy. No CMV disease was documented in the group of patients receiving ALA therapy as induction therapy. CMV viremia occurred in three patients in the acyclovir group (14.3%) and in one patient in the ganciclovir group (5%). Among renal transplant recipients only, 1 of 35 patients developed CMV disease (2.9%) and no case of CMV disease was documented in patients treated with orally administered ganciclovir. All six patients receiving two courses of ALA therapy each were free of CMV disease. Toxicity of the regimen was minimal, and antiviral resistance did not develop. Conclusions. Preemptive antiviral therapy with intravenously administered ganciclovir during ALA therapy and then orally administered ganciclovir for 3 to 4 months provides virtually complete protection against the excessive rate of CMV disease that occurs in CMV seropositive allograft recipients receiving ALA therapy.

Fibrosing cholestatic hepatitis (FCH) has recently been described after solid organ transplantation in patients with hepatitis C virus (HCV) infection. Typically, FCH is characterized by an ominous clinical course leading to progressive hepatic failure and death if liver transplantation is not performed. Two HCV-infected patients underwent cadaveric renal transplantation for end-stage renal disease resulting from membranous nephropathy and diabetic nephropathy. The time intervals between transplantation and the biopsy diagnosis of FCH for the two patients were 7 months and 10 years. Both patients presented with jaundice, hyperbilirubinemia, and mild-to-moderate elevations in serum aspartate aminotransferase. One patient was also found to have type II mixed cryoglobulinemia. Interferon-α therapy was begun after a diagnosis of FCH was established by liver biopsy. Liver test abnormalities normalized rapidly. When cholestatic hepatic deterioration develops in an HCV-infected organ allograft recipient, the diagnosis of FCH should be considered and a liver biopsy performed. Our observations indicate that FCH can respond to antiviral therapy.

We conducted a Phase I clinical trial investigating the biologic activity of vaccination with irradiated autologous melanoma cells engineered to secrete human granulocyte–macrophage colony-stimulating factor in patients with metastatic melanoma. Immunization sites were intensely infiltrated with T lymphocytes, dendritic cells, macrophages, and eosinophils in all 21 evaluable patients. Although metastatic lesions resected before vaccination were minimally infiltrated with cells of the immune system in all patients, metastatic lesions resected after vaccination were densely infiltrated with T lymphocytes and plasma cells and showed extensive tumor destruction (at least 80%), fibrosis, and edema in 11 of 16 patients examined. Antimelanoma cytotoxic T cell and antibody responses were associated with tumor destruction. These results demonstrate that vaccination with irradiated autologous melanoma cells engineered to secrete granulocyte–macrophage colony-stimulating factor stimulates potent antitumor immunity in humans with metastatic melanoma.

Background. Optimizing therapeutic monoclonal antibody (mAb) depends on the incorporation of the necessary effector functions and the development of hypoantigenic humanized antibodies by genetic engineering, which then need to be tested in appropriate preclinical trials. Methods. Constructs of humanized OKT4A containing the complementarity-determining region (CDR) of murine OKT4A and the framework and constant regions of human light (κ) and heavy chains (IgG1 and IgG4) were prepared and tested in cynomolgus monkeys who received a renal allograft. A prophylactic course of CDR-OKT4A/human (h) IgG1 or CDR-OKT4A/ hIgG4, either as high-dose single bolus (10 mglkg) or as low-dose multiple infusion (1 mglkg for 12 days) was given, and the effects on graft survival, immunohistology, circulating cells, and lymph node cells were assessed. Results. The IgG1 isotype induced coating of T cells, modulation of surface CD4 molecules, and profound depletion of CD4 + lymphocytes in peripheral blood, which persisted as long as the animals were followed (up to 7 weeks). The IgG4 isotype induced only cell coating without cell clearance or modulation. In lymph nodes, coating of lymphocytes (approximately 60%) was seen with both isotypes in the earliest sample (6 hr). After 2 days, significant depletion of lymph node CD4 cells was evident, with a decrease in the CD4 to CD8 ratio in the IgG1-treated group; no depletion occurred in the IgG4 group. The emigration of CD4 + cells into the allograft was significantly delayed in the CDR-OKT4A/hIgG1-treated animals when compared with the CDR-OKT4A-hIgG4 group as judged by immunocytochemistry 123.8±13.2 days vs. 7.4±1.5 days, P

BACKGROUND Combined kidney-pancreas transplantation (CKPT) with its associated euglycemia has been shown to prevent or reduce recurrent diabetic nephropathy in the renal allograft. There has been no evaluation of residual native kidney function after CKPT. The purpose of this study was to determine whether native kidney function may be present in diabetic recipients years after CKPT. METHODS Between 1986 and 1992, 37 patients with type 1 insulin-dependent diabetes mellitus with renal failure underwent CKPT. In each case, a single native nephrectomy was performed. We studied 16 patients who had continuing renal and pancreas function more than 4 years after CKPT. Fourteen diabetics with a functioning renal allograft but no pancreas function were used as a control group. Simultaneous renal scans (technetium-99m diethylenetriamine pentaacetic acid) of the native and transplanted kidneys were obtained with a dual-head scintillation camera. Total glomerular filtration rate (GFR) was determined from the rate of clearance of the tracer from the extracellular space measured for 2 hr with an ambulatory renal monitor. RESULTS The study groups had similar pretransplant characteristics. At the time of the study, the mean serum creatinine level was not significantly different in the CKPT and control groups (1.7+/-0.7 vs. 1.5+/-0.3 mg/dl, respectively). In the CKPT and control groups, total GFRs were 70.1+/-33 vs. 72.1+/-16.5 ml/min (NS), allograft GFRs were 63+/-34.2 vs. 70.4+/-16 ml/min (NS), and native kidney GFRs were 7.1+/-7.2 vs. 1.7+/-1.9 ml/min (P < 0.05), respectively. In both groups, there was a significant correlation between total GFR and allograft GFR (P < 0.001), but not between total GFR and native kidney GFR. Significant single native kidney GFR (more than 8 ml/min) was found in 7/16 (44%) patients in the CKPT group, but in none of the controls. CONCLUSIONS These results suggest that residual native kidney function can be present and contribute moderately to total GFR after CKPT. Euglycemia after CKPT may have a protective role in native kidneys.

Background. Hepatic artery thrombosis (HAT) remains a devastating complication after liver transplantation. Various factors have been implicated in the pathogenesis of HAT, such as clotting abnormalities, increased hematocrit, and technical complications, but the role of anticardiolipin antibodies has not been evaluated. We investigated the possible association between HAT and anticardiolipin antibodies in adult patients who underwent liver transplantation. Methods. Seven patients with HAT after orthotopic liver transplantation, 28 liver recipients without HAT, and 35 normal blood donors were evaluated. Determination of IgM and IgG anticardiolipin antibodies was performed by enzyme-linked immunosorbent assay using pretransplant serum from all allograft recipients. Clinical information was obtained from chart review. Fisher's exact test and Wilcoxon rank sum test were used for statistical analysis, and all P-values were two-tailed. Results. Overall, 22 of 35 (63%) liver recipients had a positive anticardiolipin antibody test (either IgG or IgM titer >4 SD from the normal controls). The test was positive in 7 liver recipients (100%) with HAT compared with 15 out of 28 patients (54%) without HAT (P=0.031). As compared with liver recipients without HAT, patients with HAT also tended to have a higher mean anticardiolipin titer of IgG and IgM and a lower pretransplant platelet count; however, these differences were not significant. Conclusions. Our findings indicate that anticardiolipin antibodies are frequently elevated in patients with liver failure and may contribute to the pathogenesis of HAT after liver transplantation. Other potential consequences of anticardiolipin antibodies in end- stage liver disease remain to be determined.

In recent years, hepatitis C virus infection has been reported to be typically associated with membranoproliferative glomerulonephritis and less frequently with membranous nephropathy. Treatment of hepatitis C with interferon-alpha can reduce viremia and improve renal disease. After liver transplantation for hepatitis C virus-associated liver failure, standard immunosuppressive protocols result in a significant increase in hepatitis C viremia. In this report we describe a patient with end-stage liver disease and biopsy-proven hepatitis C-associated glomerulonephritis who underwent liver transplantation. Within 1 month after transplantation, he developed a severe nephrotic syndrome that paralleled a marked increase in viremia. We discuss the possible pathogenic relationship between hepatitis C virus infection and the nephrotic syndrome that followed liver transplantation.