Nina Tolkoff-Rubin

HLA-Mismatched Renal Transplantation without Maintenance Immunosuppression

Five patients with end-stage renal disease received combined bone marrow and kidney transplants from HLA single-haplotype mismatched living related donors, with the use of a nonmyeloablative preparative regimen. Transient chimerism and reversible capillary leak syndrome developed in all recipients. Irreversible humoral rejection occurred in one patient. In the other four recipients, it was possible to discontinue all immunosuppressive therapy 9 to 14 months after the transplantation, and renal function has remained stable for 2.0 to 5.3 years since transplantation. The T cells from these four recipients, tested in vitro, showed donor-specific unresponsiveness and in specimens from allograft biopsies, obtained after withdrawal of immunosuppressive therapy, there were high levels of P3 (FOXP3) messenger RNA (mRNA) but not granzyme B mRNA.

Combined histocompatibility leukocyte antigen-matched donor bone marrow and renal transplantation for multiple myeloma with end stage renal disease : The induction of allograft tolerance through mixed lymphohematopoietic chimerism

BACKGROUND Experimental and clinical evidence has demonstrated that the establishment of allogeneic chimerism after bone marrow transplantation may provide donor-specific tolerance for solid organ allografts. METHODS Based on the preliminary results of a clinical trial using nonmyeloablative preparative therapy for the induction of mixed lymphohematopoietic chimerism, we treated a 55-year-old woman with end stage renal disease secondary to multiple myeloma with a combined histocompatibility leukocyte antigen-matched bone marrow and renal transplant after conditioning with cyclophosphamide, antithymocyte globulin, and thymic irradiation. RESULTS The posttransplant course was notable for early normalization of renal function, the absence of acute graft-versus-host disease, and the establishment of mixed lymphohematopoietic chimerism. Cyclosporine, which was the only posttransplant immunosuppressive therapy, was tapered and discontinued on day +73 posttransplant. No rejection episodes occurred, and renal function remains normal on day + 170 posttransplant (14 weeks after discontinuing cyclosporine). Although there is presently no evidence of donor hematopoiesis, there is evidence of an ongoing antitumor response with a recent staging evaluation showing no measurable urine kappa light chains. The patient remains clinically well and is off all immunosuppressive therapy. CONCLUSION This is the first report of the deliberate induction of mixed lymphohematopoietic chimerism after a nonmyeloablative preparative regimen to treat a hematological malignancy and to provide allotolerance for a solid organ transplant.

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.

Treatment of acute renal allograft rejection with OKT3 monoclonal antibody.

Eight cadaver donor renal allograft recipients, who had received azathioprine and prednisone from the day of transplantation, were treated with OKT3 monoclonal antibody (reactive with all mature peripheral blood T cells) at the time of diagnosis of acute rejection. In all cases, loss of essentially all detectable peripheral blood OKT3-reactive cells was noted within minutes after the initial 1- to 5-mg i.v. infusion. Chills and fever invariably occurred following the first or second infusion of monoclonal antibody, but were not noted during the subsequent, 10- to 20-day course of therapy, suggesting rapid cell lysis as the etiology of this toxicity. The established rejection episode was reversed in all cases within 2 to 7 days without addition of any therapy other than OKT3 antibody and despite continued lowering of the steroid dosages. During the subsequent 3- to 12-month follow-up period, further rejection episodes occurred in five of these patients, two of these were irreversible with conventional therapy so that six of the eight allografts continue with excellent renal function. These preliminary observations suggest that homogeneity, limited dosage requirements, and ease of in vitro monitoring of dosage effects should markedly simplify the use of monoclonal antibody to T cell populations in human allograft recipients. This second generation of antilymphocyte preparations offers the potential for not only increased effectiveness but also the possibility of manipulating specific T cell subsets.

Infection in the renal transplant recipient

The incidence of infection in the renal transplant patient is directly related to the net immunosuppressive effect achieved and the duration of time over which this therapy is administered. A second major factor in the causation of infections in this population is the nosocomial hazards to which these patients are exposed, ranging from invasive instrumentation to environmental contamination with Aspergillus species, Legionella pneumophila, Pseudomonas aeruginosa and other microbial pathogens. Careful surveillance is necessary to identify and eliminate such nosocomial sources of infection. The major types of infection observed can be categorized according to the time period post-transplant in which they occur: postsurgical bacterial infection in the first month after transplantation; opportunistic infection, with cytomegalovirus playing a major role, and transplant pyelonephritis in the period one to four months post-transplant; and a mixture of conventional and opportunistic infections in the last post-transplant period. Conventional infection in this late period occurs primarily in patients with good renal function who are receiving minimal immunosuppressive therapy; opportunistic infection occurs primarily in patients with poor renal function who are receiving higher levels of immunosuppression.

Controlled clinical trial of prophylactic human-leukocyte interferon in renal transplantation. Effects on cytomegalovirus and herpes simplex virus infections.

A double-blind, placebo-controlled trial of interferon prophylaxis against viral infections was conducted in renal-transplant recipients receiving standard immunosuprressive therapy with or without antithymocyte globulin. Interferon was administered for six weeks, beginning on the day of transplantation. Cytomegalovirus excretion began earlier and viremia was more frequent in placebo-treated than in interferon-treated patients. Cytomegalovirus viremia correlated with clinical syndromes was more frequent in recipients of antithymocyte globulin. In contrast, neither interferon nor antithymocyte globulin altered excretion of herpes simplex virus. Reversible leukopenia and thrombocytopenia occurred in seven interferon recipients. Patient and graft survival were comparable in interferon and placebo groups. There preliminary results suggest that a six-week course of prophylactic interferon delays shedding of cytomegalovirus and decreases the incidence of viremia after transplantation. In contrast, antithymocyte globulin appears to increase the severity of infection from cytomegalovirus among these patients.

Myeloma Responses and Tolerance Following Combined Kidney and Nonmyeloablative Marrow Transplantation: In Vivo and In Vitro Analyses

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.

Glomerulopathy Associated with Cytomegalovirus Viremia in Renal Allografts

Abstract We investigated the relation between cytomegalovirus (CMV) infection and renal-allograft dysfunction in 14 patients. In seven instances (including two successive transplants in one patient), allograft dysfunction occurred during clinically manifest, viremic CMV infection. In five of these, biopsies revealed little or no tubulointerstitial change but a distinctive, diffuse glomerulopathy characterized by enlargement or necrosis of endothelial cells and accumulation of mononuclear cells and fibrillar material in glomerular capillaries. Two of these allografts recovered their function, both with cessation of high-dose immunosuppression. Biopsies in the other 10 patients revealed predominantly tubulointerstitial changes typical of cellular rejection, and most of these patients did not have viremia. One additional patient, studied prospectively, manifested both forms of allograft injury: tubulointerstitial changes occurring two weeks after transplantation and responding to increased immunosuppression,...

Posttransplant diabetes mellitus in liver transplant recipients: risk factors, temporal relationship with hepatitis C virus allograft hepatitis, and impact on mortality

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 <0.0001). The cumulative mortality in HCV (+) PTDM (+) versus HCV (+) PTDM (-) patients was 56% vs. 14% (P =0.001). In HCV (+) patients with PTDM, we could identify two groups based on the temporal relationship between the allograft hepatitis and the onset of PTDM: 13 patients developed PTDM either before or in the absence of hepatitis (group A), and 12 concurrently with the diagnosis of hepatitis (group B). In gr. B, 11 of 12 patients received antiviral therapy. Normalization of liver function tests with improvement in viremia was achieved in 4 of 11 patients, who also demonstrated a marked improvement in their glycemic control. Conclusion. We found a high prevalence of PTDM in HCV (+) recipients. PTDM after OLT was associated with significantly increased mortality. HCV infection and methylprednisolone boluses were found to be independent risk factors for the development of PTDM. In approximately half of the HCV (+) patients with PTDM, the onset of PTDM was related to the recurrence of allograft hepatitis. Improvement in glycemic control was achieved in the patients who responded to antiviral therapy.

Preemptive Ganciclovir Therapy To Prevent Cytomegalovirus Disease in Cytomegalovirus Antibody-Positive Renal Transplant Recipients: A Randomized Controlled Trial

Cytomegalovirus is the most important infectious cause of complications and death in organ transplant recipients. The three major consequences of infection with this virus are cytomegalovirus disease; superinfection with opportunistic pathogens resulting from host defects caused by the virus; and allograft injury [1, 2]. The interaction of the following three factors determines whether cytomegalovirus disease develops in a transplant recipient infected with cytomegalovirus: 1) whether the donor and donor organ or the recipient, or both, harbor latent virus that can be reactivated after transplant [1]; 2) whether the transplant recipient can mount an immune response to the virus [both cellular and humoral]; and 3) the type of immunosuppressive therapy administered after transplant [4]. Transplant recipients in whom primary infection develops at the time of transplant (donor: cytomegalovirus antibody positive; recipient: cytomegalovirus antibody negative) or who test positive for cytomegalovirus antibody before transplantation and require antilymphocyte antibody therapy after transplantation have a greater than 50% attack rate of cytomegalovirus disease [4]. Each risk group accounts for 10% to 20% of all transplant recipients. Preventing cytomegalovirus disease in these two patient populations is a priority for transplant physicians. Cytomegalovirus disease has been prevented by prolonged administration (3 to 4 months) of antiviral therapies such as acyclovir [5], anticytomegalovirus hyperimmune globulin [6], or the combination of these two therapies [7]. These prophylactic strategies reduce the attack rate of cytomegalovirus disease, but this benefit is attenuated in patients who receive antilymphocyte antibody therapy [1]. Because the risk for developing cytomegalovirus disease depends on the type of immunosuppression administered after transplantation, we have proposed an alternative approach to preventing cytomegalovirus disease. This approach targets patients at greatest risk for cytomegalovirus disease for treatment with the most potent anticytomegalovirus therapy available. The antiviral therapy is administered when the risk is greatest (for example, during treatment with antilymphocyte antibodies). To distinguish this approach from preventive strategies used in all patients (nontargeted prophylaxis), we introduced the term preemptive therapy [8]. Preliminary studies suggested that ganciclovir administered as preemptive therapy to patients receiving antilymphocyte antibodies might reduce the attack rate of cytomegalovirus disease in transplant recipients who are positive for cytomegalovirus antibody. This multiinstitutional, randomized clinical trial was designed to assess the efficacy and safety of preemptive ganciclovir therapy in preventing cytomegalovirus disease in transplant recipients who are positive for cytomegalovirus antibody and who are receiving antilymphocyte antibodies. Methods Patients Consecutive renal transplant recipients who tested positive for cytomegalovirus antibody and who received a kidney from cytomegalovirus antibody-positive or antibody-negative donors at Albany Medical Center, Emory University Hospital, Massachusetts General Hospital, New England Medical Center, University of Chicago Medical Center, and Yale-New Haven Hospital were eligible for enrollment in the study. Patients were randomly assigned to receive either preemptive ganciclovir or no ganciclovir every day that antilymphocyte antibody therapy (muromonab-CD3 [OKT3], antithymocyte globulin, or antilymphocyte globulin) was administered. Separate randomization lists were used in each transplantation center; within each center, separate randomization lists were used for cadaveric and living, related donors. The investigators at each site knew which patients received the study drug and which patients received no anticytomegalovirus therapy. The cytomegalovirus antibody status of donors and recipients was determined using enzyme immunoassay on recipient serum obtained before transplantation and on donor serum obtained before transfusion. Patients were excluded from the study if they were younger than 20 years of age, were pregnant, had received another organ in addition to a kidney, had received any anticytomegalovirus therapy (defined as more than 1.2 g of acyclovir per day, unselected immune globulin, or cytomegalovirus hyperimmune globulin), or refused to give consent. The committees on human experimentation at each institution approved the study. All study participants were observed for the following outcomes during the 6 months after they received antilymphocyte antibodies: 1) cytomegalovirus disease; 2) other infectious diseases; 3) and noninfectious diagnoses. Allograft function 6 months after administration of antilymphocyte antibody was recorded as either present (for those not requiring dialysis) or absent (for those receiving dialysis). Serum creatinine levels were recorded for all patients with functioning allografts 6 months after antilymphocyte antibody therapy. Patients were evaluated for cytomegalovirus viremia and disease in three ways. First, the virology laboratory at each program tried to isolate cytomegalovirus from buffy-coat specimens at least every month using either centrifugation culture [9], conventional culture techniques [10], or both. Second, buffy-coat specimens were cultured for cytomegalovirus when any of the following signs, symptoms, or laboratory abnormalities were present: temperature greater than 38 C, leukocyte count less than 3.0 109 cells/L, dyspnea, abdominal pain, or gastrointestinal bleeding. Third, a biopsy specimen was obtained from any abnormal site, and all biopsy specimens were examined histologically for the presence of characteristic cytomegalovirus inclusion bodies and uptake of immunofluorescent-labeled anticytomegalovirus antibodies. Patients with other signs or symptoms suggesting infection (such as cough, headache, diarrhea, allograft discomfort, or dysuria) were evaluated using standard diagnostic protocols. At all study sites, the diagnostic protocol included at least a chest radiograph, leukocyte count, renal and liver function tests, two sets of blood cultures, urinalysis, urine culture, and cultures from other potential sites of infection. For patients who did not survive the 6-month observation period, data on the cause and date of death were collected. Definition of Cytomegalovirus Disease Cytomegalovirus disease was defined as either the cytomegalovirus syndrome or tissue-invasive disease developing within 6 months of antilymphocyte antibody therapy or within 1 month of discontinuing immunosuppression after allograft loss. The cytomegalovirus syndrome was diagnosed when both virologic and clinical criteria were met within a 7-day period. Virologic criteria were fulfilled when cytomegalovirus was isolated from a buffy-coat specimen or bronchoalveolar fluid. Clinical criteria were fulfilled when patients had a temperature higher than 38 C [without antipyretic agents] for 3 or more consecutive days and within 7 days of two or more of the following: 1) leukopenia [leukocyte count <3.0 109 cells/L on two consecutive measurements after stopping azathioprine therapy]; 2) hepatitis [serum alanine aminotransferase >1.5 times the upper limit of normal, without serologic evidence of active hepatitis B or hepatitis C virus]; 3) atypical lymphocytosis [more than 20% of leukocytes]; and 4) pneumonitis (an abnormal result on chest radiograph and no alternative explanation [including absence of Pneumocystis carinii in respiratory secretions]). These criteria were modified slightly from those used by other researchers [6] because isolation of cytomegalovirus from buffy-coat specimens or bronchoalveolar fluid predicts presence of cytomegalovirus disease, whereas isolation of cytomegalovirus from urine and saliva may not [11]. Tissue-invasive cytomegalovirus disease was diagnosed histopathologically by showing the presence of inclusion bodies characteristic of cytomegalovirus or by an immunochemical stain positive for cytomegalovirus antigens in a biopsy specimen from a lesion or an abnormal site (gastrointestinal tract, lung, or liver). The investigator at each study site made decisions about treating cytomegalovirus disease. Study Drug Administration The study drug was given daily (or according to the schedule in Table 1 only when the patient already had intravenous access for administration of antilymphocyte antibodies. The study medication was started within 24 hours of the first dose of each course of antilymphocyte antibodies. Patients receiving the study drug were given ganciclovir infusions based on daily serum creatinine concentrations (Table 1). Table 1. Ganciclovir Dosage Schedule according to Daily Serum Creatinine Concentration Leukocyte count, platelet count, and serum creatinine concentration were monitored daily during antilymphocyte antibody therapy in patients who received preemptive ganciclovir therapy and in controls. Sample Size and Statistical Analysis In the primary analysis, the proportion of patients with cytomegalovirus disease in the two study groups were compared. On the basis of our previous study [4], we assumed that cytomegalovirus disease would develop in 60% of controls. We wanted to enroll 48 patients who could be evaluated in each group (for a total of 96) to detect a decrease in the proportion of patients with cytomegalovirus disease from 60% (in controls) to 30% or less (in patients receiving preemptive ganciclovir therapy); that is, we predicted that preemptive ganciclovir treatment would yield a 50% lower attack rate (using a two-sided test, = 0.05 and power = 0.8). Initial analyses were done on an intention-to-treat basis and included all eligible patients. We defined the primary outcome and potential predictors of cytomegalovirus disease before beginning the study. At the end of therapy and follow-up, the study nurse purged all study si