Shamila Mauiyyedi

Identification of the CD4+ T cell as a major pathogenic factor in ischemic acute renal failure

Leukocytes have been implicated in the pathogenesis of ischemic acute renal failure (ARF), but the roles of the individual cell types involved are largely unknown. Recent indirect evidence suggests that T cells may play an important role in a murine model of ARF. In the current study, we found that mice deficient in T cells (nu/nu mice) are both functionally and structurally protected from postischemic renal injury. Reconstitution of nu/nu mice with wild-type T cells restored postischemic injury. We then analyzed the contribution of the individual T cell subsets to postischemic injury and found that mice deficient in CD4(+) T cells, but not mice deficient in CD8(+) T cells, were significantly protected from ARF. Direct evidence for a pathophysiologic role of the CD4(+) T cell was obtained when reconstitution of CD4-deficient mice with wild-type CD4(+) T cells restored postischemic injury. In addition, adoptive transfers of CD4(+) T cells lacking either the costimulatory molecule CD28 or the ability to produce IFN-gamma were inadequate to restore injury phenotype. These results demonstrate that the CD4(+) T cell is an important mediator of ischemic ARF, and targeting this cell may yield novel therapies.

Acute humoral rejection in renal allograft recipients: I. Incidence, serology and clinical characteristics.

Background. Acute rejection (AR) associated with de novo production of donor-specific antibodies (DSA) is a clinicopathological entity that carries a poor prognosis (acute humoral rejection, AHR). The aim of this study was to determine the incidence and clinical characteristics of AHR in renal allograft recipients, and to further analyze the antibodies involved. Methods. During a 4-year period, 232 renal transplants (Tx) were performed at our institution. Assays for DSA included T and B cell cytotoxic and/or flow cytometric cross-matches and cytotoxic antibody screens (PRA). C4d complement staining was performed on frozen biopsy tissue. Results. A total of 81 patients (35%) suffered at least one episode of AR within the first 3 months: 51 had steroid-insensitive AR whereas the remaining 30 had steroid-sensitive AR. No DSA were found in patients with steroid-sensitive AR. In contrast, circulating DSA were found in 19/51 patients (37%) with steroid-insensitive AR, and widespread C4d deposits in peritubular capillaries were present in 18 of these 19 (95%). In at least three cases, antibodies were against donor HLA class II antigens. DSA were not found in the remaining 32 patients but C4d staining was positive in 2 of 32. The DSA/C4d positive (n=18) and DSA/C4d negative (n=30) groups differed in pre-Tx PRA levels, percentage of re-Tx patients, refractoriness to antilymphocyte therapy, and outcome. Plasmapheresis and tacrolimus-mycophenolate mofetil rescue reversed rejection in 9 of 10 recipients with refractory AHR. Conclusion. More than one-third of the patients with steroid-insensitive AR had evidence of AHR, often resistant to antilymphocyte therapy. Most cases (95%) with DSA at the time of rejection had widespread C4d deposits in peritubular capillaries, suggesting a pathogenic role of the circulating alloantibody. Combined DSA testing and C4d staining provides a useful approach for the early diagnosis of AHR, a condition that often necessitates a more intensive therapeutic rescue regimen.

Plasma exchange and tacrolimus-mycophenolate rescue for acute humoral rejection in kidney transplantation

BACKGROUND Acute renal allograft rejection associated with the development of donor-specific alloantibody (acute humoral rejection, AHR) typically carries a poor prognosis. The best treatment of this condition remains undefined. METHODS During a 14-month period, 73 renal transplants were performed. During the first postoperative month, five recipients (6.8%) with AHR were identified. The diagnosis was based on: (1) evidence of severe rejection, resistant to steroid and antilymphocyte therapy; (2) typical pathologic features; and (3) demonstration of donor-specific alloantibody (DSA) in recipients serum at the time of rejection. Pretransplant donor-specific T- and B-cell cross-matches were negative. RESULTS Plasma exchange (PE, four to seven treatments per patient) significantly decreased circulating DSA to almost pretransplant levels in four of five patients, and improvement in renal function occurred in all patients. One patient had recurrent renal dysfunction in the setting of an increase in circulating DSA. A second series of five PE treatments decreased DSA and reversed the rejection episode. Rescue therapy with tacrolimus (initial mean dose: 0.14+/-0.32 mg/kg/day) and mycophenolate mofetil (2 g/day) was used in five of five and four of five patients, respectively. With a mean follow-up of 19.6+/-5.6 months, patient and allograft survival are 100%. Renal function remains excellent with a mean current serum creatinine of 1.2+/-0.3 mg/dl. (range: 0.9-1.8 mg/dl). CONCLUSIONS Our findings suggest that a therapeutic approach combining PE and tacrolimus-mycophenolate mofetil rescue has the potential to improve the outcome of AHR.

Long-term outcome and alloantibody production in a non-myeloablative regimen for induction of renal allograft tolerance

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.

Overlapping pathways to transplant glomerulopathy: chronic humoral rejection, hepatitis C infection, and thrombotic microangiopathy

Transplant glomerulopathy (TG) has received much attention in recent years as a symptom of chronic humoral rejection; however, many cases lack C4d deposition and/or circulating donor-specific antibodies (DSAs). To determine the contribution of other causes, we studied 209 consecutive renal allograft indication biopsies for chronic allograft dysfunction, of which 25 met the pathological criteria of TG. Three partially overlapping etiologies accounted for 21 (84%) cases: C4d-positive (48%), hepatitis C-positive (36%), and thrombotic microangiopathy (TMA)-positive (32%) TG. The majority of patients with confirmed TMA were also hepatitis C positive, and the majority of hepatitis C-positive patients had TMA. DSAs were significantly associated with C4d-positive but not with hepatitis C-positive TG. The prevalence of hepatitis C was significantly higher in the TG group than in 29 control patients. Within the TG cohort, those who were hepatitis C-positive developed allograft failure significantly earlier than hepatitis C-negative patients. Thus, TG is not a specific diagnosis but a pattern of pathological injury involving three major overlapping pathways. It is important to distinguish these mechanisms, as they may have different prognostic and therapeutic implications.

BK virus-associated nephropathy in sirolimus-treated renal transplant patients : Incidence, course, and clinical outcomes

Background. Because the course of polyoma virus–associated nephropathy (PVAN) has not been evaluated in a large cohort of patients receiving sirolimus (SRL)-based regimens, we have herein presented the incidence, clinical characteristics, and outcomes of 378 renal transplant recipients treated with SRL-based immunosuppression. Methods. This retrospective single center study evaluated 344 kidney alone (KTX) and 34 simultaneous pancreas-kidney (SPK) transplantations performed between June 2000 and December 2004. Results. At a mean follow-up of 43.3 months, six kidney (1.7%) and three kidney-pancreas (9.0%) transplanted patients displayed biopsy-proven PVAN. The mean time to diagnosis after transplantation was 18.2 months (range: 3.5–31.1 months), with a higher incidence among patients exposed (4.23%) versus not exposed to rabbit antithymocyte globulin (rATG; 0.53%; P=0.019) or SPK (9.0%) versus KTX (1.7%) recipients (odds ratio: 5.43; confidence interval: 1.29–22.8; P=0.038). Despite treatment with cidofovir, reduced immunosuppression and maintenance therapy with no agents other than SRL (C0=10.2±2.7 ng/dL) plus modest doses of prednisone (≤5 mg), five patients (55.5%) experienced renal allograft failure. No rejection episodes were documented during the PVAN treatment and pancreatic function continued to be excellent among the SPK patients. Conclusions. Patients treated with SRL-based immunosuppression showed an incidence at the lower end of the range described with various other contemporaneous immunosuppressive regimens and with other cohorts not undergoing BK virus polymerase chain reaction surveillance. Exposure to rATG and SPK transplantation represented risk factors for the occurrence of PVAN, which showed a pernicious course despite withdrawal of calcineurin antagonists and/or mycophenolate mofetil.

Posttransplant lymphoproliferative disorder associated with an Epstein-Barr-related virus in cynomolgus monkeys.

Background. Human posttransplant lymphoproliferative disorder (PTLD) has been shown to be associated with Epstein-Barr virus (EBV) infection. Primate animal models of PTLD and the use of molecular markers in its diagnosis have not been reported. This study was designed to evaluate the frequency, pathology, and molecular characteristics of PTLD in cynomolgus kidney allograft recipients. Methods. Over a 5-year period (January 1995 to November 2000), 160 primate renal transplants were performed at the Massachusetts General Hospital (MGH). Of these, all cases (n=9) that developed PTLD were included. H&E stained paraffin sections of all available tissue samples from the cases were evaluated for the presence of PTLD. Immunoperoxidase staining for T cells (CD3), B cells (CD20), kappa and lambda light chains as well as EBV nuclear antigens (EBNA2) and latent membrane proteins (EBV LMP-1) was done on paraffin sections using standard immunohistochemical (IHC) methods. In situ hybridization for EBV encoded RNA (EBER) was performed in all tissue samples with atypical lymphoid proliferations, using a novel EBER nucleotide probe based on consensus gene sequences from EBV and the related herpes lymphocryptoviruses (LCV) infecting baboons and rhesus macaques. Results. Of 160 consecutive primate renal transplants performed at MGH, 5.6% developed PTLD 28–103 days after transplantation. In all cases, the lymph nodes were involved and effaced by an atypical polymorphous lymphoid proliferation of EBER+ B cells, diagnostic for PTLD. Focal staining for EBNA-2 was noted in tumor cells. In 67% (six of nine) the PTLD infiltrates were present in extra nodal sites, notably liver (56%), lung (44%), heart (44%), renal allograft (44%), and native kidney (22%). The spleen was involved by PTLD in all four animals that had not undergone a pretransplant splenectomy. The PTLD morphology was similar in all cases and predominantly of the polymorphous type, however, some of these showed areas that appeared minimally polymorphous. No cases of monomorphic PTLD were seen. Conclusions. By in situ hybridization, expression of the RNA product, homologous for EBV-encoded RNA (EBER) was identified in the PTLD tumor cells of all cases, indicating latent primate EBV- related infection. This report identifies a novel animal model of EBV associated PTLD in the setting of kidney transplantation, with valuable implications for managing and understanding human PTLD and oncogenesis.

Association of natural killer cell depletion with induction of mixed chimerism and allograft tolerance in non-human primates.

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. METHOD 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. <5 days). 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.

Correlation of Biochemical and Hematological Changes with Graft Failure Following Pig Heart and Kidney Transplantation in Baboons

We have explored biochemical and hematologic parameters that might indicate acute humoral xenograft rejection (AHXR) following pig organ transplantation in baboons.

Induction of molecular chimerism by gene therapy prevents antibody-mediated heart transplant rejection

In order for xenotransplantation to become a clinical reality, and fulfill its promise of overcoming shortages of human organs and tissues, rejection mediated by the hosts immune system must first be overcome. In primates, preformed natural antibodies that bind the carbohydrate antigen Galα1-3Galβ1-4GlcNAc-R (αGal), which is synthesized by UDP galactose:ß-D-galactosyl-1,4-N-acetyl-D-glucosaminide α(1-3)galactosyltransferase (E.C. 2.4.1.151) or simply αGT, mediate rigorous rejection of transplanted pig organs and tissues. In αGT knockout mice (GT0 mice), which like humans contain in their serum antibodies that bind αGal, expression of a retrovirally transduced αGT in bone marrow-derived cells is sufficient to prevent production of αGal-reactive antibodies. Here, we demonstrate that reconstitution of lethally irradiated GT0 mice with αGT-transduced bone marrow cells from GT0 littermates prevents antibody-mediated rejection of cardiac transplants from wild-type mice. These data suggest that gene therapy can be used to induce immunological tolerance to defined antigens and thereby overcome transplant rejection.