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AIM To compare laparoscopic and open living donor nephrectomy, based on the results from a single center during a decade. METHODS This is a retrospective review of all living donor nephrectomies performed at the Massachusetts General Hospital, Harvard Medical School, Boston, between 1/1998 - 12/2009. Overall there were 490 living donors, with 279 undergoing laparoscopic living donor nephrectomy (LLDN) and 211 undergoing open donor nephrectomy (OLDN). Demographic data, operating room time, the effect of the learning curve, the number of conversions from laparoscopic to open surgery, donor preoperative glomerular filtration rate and creatinine (Cr), donor and recipient postoperative Cr, delayed graft function and donor complications were analyzed. Statistical analysis was performed. RESULTS Overall there was no statistically significant difference between the LLDN and the OLDN groups regarding operating time, donor preoperative renal function, donor and recipient postoperative kidney function, delayed graft function or the incidence of major complications. When the last 100 laparoscopic cases were analyzed, there was a statistically significant difference regarding operating time in favor of the LLDN, pointing out the importance of the learning curve. Furthermore, another significant difference between the two groups was the decreased length of stay for the LLDN (2.87 d for LLDN vs 3.6 d for OLDN). CONCLUSION Recognizing the importance of the learning curve, this paper provides evidence that LLDN has a safety profile comparable to OLDN and decreased length of stay for the donor.

Background: Lymphatic leak and lymphocele are well-known complications after kidney transplantation.Objective: To determine the incidence of lymphatic complications in recipients of living donor kidneys.Methods: Among 642 kidney transplants performed between 1999 and 2007, the incidence of lymphatic complicationswas retrospectively analyzed in recipients of living donor kidneys procured by laparoscopic nephrectomy(LP, n=218) or by open nephrectomy (OP, n=127) and deceased donor kidneys (DD, n=297). A Jackson-Pratt drain was placed in the retroperitoneal space in all recipients and was maintained until the output became less than 30 mL/day.Results: Although the incidence of symptomatic lymphocele, which required therapeutic intervention, was comparable in all groups, the duration of mean±SD drain placement was significantly longer in the LP group—8.6±2.7 days compared to 5.6±1.2 days in the OP group and 5.4±0.7 days in the DD group (p

Background: Hepatitis C (HCV) is the most common indication for liver transplantation in the US. Objective: Since steroids are the major stimulus of viral replication, we postulated that steroid-free immunosuppression might be a safer approach. Methods: From January 1995 to October 2002, we used steroid plus calcineurin inhibitor (CNI) immunosuppression after liver transplantation for HCV (steroid group, n=81). From October 2002 to June 2007, rabbit antithymocyte globulin (RATG) induction, followed by CNI and azathioprine (RATG group, n=73) was utilized. Results: There were no differences in 1- and 3-year patient/allograft survival rates. The incidence of acute rejection rate (19% vs. 28%), of biopsy-proven HCV recurrence (70% vs. 75%), and chronic rejection (6% vs. 9%) were comparable. The mean time to develop recurrent HCV was significantly longer in the RATG group (16.2 vs. 9.2 months, p=0.008). The incidence of severe portal fibrosis appears to be lower in RATG group compared to the steroid group; 14% vs. 4% (p=0.07). Conclusions: RATG induction is safe and effective after liver transplantation for HCV, but has no impact on the incidence of HCV recurrence and patient/allograft survival. However, a significant delay in time to HCV recurrence and a trend toward less rejection and portal fibrosis was observed.

These studies test whether allograft rejection can be blocked by interference with leukocyte adhesion, using a murine IgG2a mAb (R6.5) reactive with monkey ICAM-1 (CD54). In 16 Cynomolgus renal allograft recipients, R6.5 was administered prophylactically as the sole immunosuppressive agent for 12 days (0.01 to 2 mg/kg/day). Survival in 14 recipients with technically successful grafts was significantly prolonged (24.2 +/- 2.4 vs 9.2 +/- 0.6 days for controls; p less than 0.001). Intercellular adhesion molecule-1 (CD54) (ICAM-1) was expressed on vascular endothelium in the kidney and other organs in the monkey in a pattern similar to that in humans. During cellular rejection in controls, ICAM-1 expression increased on endothelial cells, infiltrating mononuclear leukocytes and tubular cells. Biopsies during R6.5 administration showed decreased T cell infiltration (CD2, CD8, CD4) compared with controls and decreased arterial endothelial inflammation. No changes occurred in circulating T cells, aside from variable coating with mIgG. In six of eight other recipients R6.5 administration (0.5 to 2 mg/kg/day for 10 days) reversed preexisting rejection that resulted from taper of Cyclosporine to subtherapeutic levels. Responding grafts showed decreased edema and hemorrhage but no consistent change in the infiltrate. At 1 h after the first dose, mouse IgG deposited primarily on the graft vascular endothelium without any change in the inflammatory infiltrate. Mouse IgG also deposited on the endothelium of normal organs without eliciting an inflammatory response and was cleared from the endothelium within 4 days. Inasmuch as the principal site of binding was the vascular endothelium, we hypothesize that the antibody blocks adhesion to graft ICAM-1 molecules on the vessels. Anti-ICAM-1 also binds to recipient cells and may interfere with Ag presentation and/or T cell interactions. Whatever the mechanism(s), these studies indicate that an anti-ICAM-1 antibody inhibits T cell mediated injury in vivo, and that ICAM-1 is a critical molecule in the pathogenesis of allograft rejection.

The 2 principal factors implicated in late kidney allograft failure are chronic rejection (also called chronic allograft nephropathy) and death of the patient with a functioning graft (mainly from cardiovascular causes). Despite lifelong immunosuppression of the recipient, immunological responses remain the leading factor in the pathogenesis of chronic rejection and both cellular and humoral immune mechanisms have been shown to play important roles. In this review, we highlight the relevance of humoral mechanisms of rejection to the pathogenesis of late allograft loss. Non immunological factors, such as donor organ quality, initial ischemic injury, calcineurin inhibitor (CNI) toxicity, hypertension, and hyperlipidemia, also contribute to progressive chronic allograft injury, but will not be reviewed in detail here. Possible strategies to stabilize or improve allograft function in patients with already established "chronic rejection/chronic allograft nephropathy" (CR/CAN) are the addition of mycophenolate mofetil (or sirolimus) with or without a reduction of cyclosporine dosage, or conversion from cyclosporine to tacrolimus. However, prospective randomized clinical trials are needed to test the efficacy of these strategies. A major current challenge for transplant physicians is to develop regimens that prevent CR/CAN, since, once established, the process typically progresses inexorably to renal allograft loss in most recipients. Evidence is now accumulating that new immunosuppressive regimens must control not only T cell but also B cell responses (i.e. limit antidonor antibody production) in order to prevent CR/CAN and improve long-term allograft survival.

Between February 1963 and December 2000, 1,627 kidney transplants were performed at the Massachusetts General Hospital. The majority (62%) were from cadaveric donors, although in recent years (1996-2000) 52% have been allografts from living donors, with an increase in living unrelated donors. The introduction of CsA and OKT3 in 1984 was associated with a significant improvement in actuarial renal allograft survival, although a persistent late attrition of allografts continues beyond the first year after transplantation. As reported in other centers, current actuarial survival for living unrelated allografts is superior to that of cadaveric allografts, and is quite similar to that observed in recipients of non-HLA identical living-related transplants. Our preliminary laparoscopic donor nephrectomy experience is encouraging as excellent allograft survival and function has been observed, with minimal morbidity associated with the procedure and a low rate of conversion to open nephrectomy. Recent changes in immunosuppressive protocols have resulted in lower early acute rejection rates, however the incidence of delayed graft function remains unchanged in cadaveric renal transplantation. The role of humoral immunity in allograft rejection has been progressively clarified and new approaches to control donor specific alloantibody production have been shown to be effective. Current clinical studies are ongoing to determine the optimal type and dose of calcineurin inhibitors beyond the first year after transplantation and to study whether avoidance of steroids is safe and feasible. Finally, an innovative tolerance induction protocol using the mixed chimerism approach has been successfully accomplished in selected patients with end-stage renal disease secondary to multiple myeloma. These encouraging observations emphasize that major changes from current immunosuppressive regimens are likely to occur over the next few years as more approaches to tolerance induction are explored clinically.

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.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.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, P0.0001). 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.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.

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.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.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.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.

In renal transplantation, chronic rejection is a major cause of late allograft loss. Recent studies indicate that a subset of chronic rejection is associated with anti-HLA donor specific antibodies (DSA) and complement C4d deposition in peritubular capillaries (PTC). Since rescue therapy with tacrolimus and mycophenolate mofetil has been found to limit antidonor B-cell responses in recipients with acute humoral rejection, we sought to determine whether a similar immunosuppressive regimen might be effective in patients with 'chronic humoral rejection'.Four renal allograft recipients with 'chronic humoral rejection' were prospectively identified. The diagnosis was based on: (1) progressive rise in serum creatinine over 12 months; (2) typical pathologic features by light microscopy (transplant arteriopathy and glomerulopathy); (3) widespread C4d deposits in PTC by immunofluorescence; (4) detection of 'de novo' DSA at the time of biopsy. Maintenance immunosuppression was CsA, prednisone and azathioprine (n=3) or prednisone and azathioprine (n=1). Rescue therapy with tacrolimus and mycophenolate mofetil was initiated in all patients, 12 hr after cyclosporine and azathioprine discontinuation.At diagnosis, the mean serum creatinine was 3.9 mg/dl (range: 3.3 to 5.4 mg/dl). DSA was an IgG directed against HLA class II (n=3) or class I (n=2), that is one patient had both anti-HLA class I and class II antibodies. Pretreatment antibody titers varied between 1:8 and 1:128. Rescue therapy was associated with a rapid and sustained decrease in antibody titers. In two patients, DSA became undetectable after 9 months and a repeat biopsy performed after 12 months revealed a decrease in C4d deposition in PTC.These results suggest that a decrease in DSA production can be induced in renal allograft recipients with 'chronic humoral rejection' by using an immunosuppressive regimen that combines tacrolimus and mycophenolate mofetil. Limitation of antidonor antibody synthesis may be important for the treatment or the prevention of chronic rejection in organ transplantation.

Although the primary treatment of symptomatic cytomegalovirus (CMV) disease in organ transplant recipients is successful in90% of individuals, relapsing disease, particularly in those with primary infection, remains an important problem. Previously, we had observed that the rate of symptomatic recurrence was60% 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.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.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.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.

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.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.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.G-CSF was well tolerated in solid organ transplant recipients. It was particularly useful in patients with CMV disease, allowing optimal ganciclovir therapy.

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.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.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.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.

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.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).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).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.

Idiopathic giant esophageal ulcers in immunocompromised patients have been described only in patients with acquired immunodeficiency syndrome. A solitary report of an idiopathic giant esophageal ulcer in an immunocompetent patient exists. We describe a case of idiopathic esophageal ulceration ultimately responsive to steroid therapy in a 31-year old immunosuppressed, human immunodeficiency virus-negative renal transplant patient. The case is described with particular reference to the evaluation, differential diagnosis, and therapeutic response to steroids. Similarities in presentation and treatment to giant esophageal ulcers in human immunodeficiency virus infection suggest an underlying immune defect as the likely cause. This is the first described case of giant esophageal ulceration responsive to steroids in an immunosuppressed human immunodeficiency virus-negative patient. This entity should be added to the differential diagnosis of esophageal ulceration in solid organ transplant recipients.

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.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.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 (microg/ml+/-SD) achieved in prophylaxis were P: 7.98+/-3.34, T: 3.03+/-2.63; and in treatment were P: 9.00+/-3.72, T: 2.65+/-1.82. Despite corrections for renal dysfunction, undialyzed patients with serum creatinine3.0 mg/dl had trough levels in excess of the population mean (T: range 3-8 microg/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, P0.01). Patients at greatest risk for relapse after treatment of CMV disease were liver transplant recipients, patients with ganciclovir-resistant viral isolates, and renal patients with six antigen MHC donor-recipient mismatches.This trial demonstrates the efficacy of a nomogram for ganciclovir dosing during renal dysfunction; reduced doses can be used for prophylaxis for undialyzed patients with renal dysfunction (1.25 mg/kg/day for Cror =3.0, 1.25 mg/kg QOD for Cror =5.0). Some groups of transplant recipients may require more intensive anti-CMV regimens.

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.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.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.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.

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.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.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.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.

Sentinel lymph node biopsy following radioisotope labeling is a recently developed, minimally invasive surgical staging procedure used in the management of primary cutaneous malignant melanoma. If histologic analysis reveals melanoma metastasis in the sentinel lymph node, completion lymphadenectomy is performed and adjuvant therapy considered. The routine pathologic assessment of the sentinel lymph node consists of bisecting the lymph node along its long axis and histologic examination of one hematoxylin and eosin-stained section of each cut surface.In this study, the authors reexamined 235 sentinel lymph nodes reported as negative for melanoma metastasis following routine histologic examination, from 94 patients with American Joint Committee on Cancer (AJCC) Stage I and II cutaneous melanoma.Deeper sections into the lymph node and immunohistochemical stains with antibodies to S-100, HMB-45, NK1C3, and MART-1 led to the identification of microscopic metastases in 11 sentinel lymph nodes from 11 patients and capsular nevi in 9 sentinel lymph nodes from 8 patients.Deeper serial sections and immunohistochemical stains detected microscopic metastases in approximately 12% of cases that would be reported as negative for metastasis by routine pathologic analysis. These techniques also allowed for the identification of capsular melanocytic nevi in the sentinel lymph nodes of 9% of patients. [See editorial on pages 551-2, this issue.]

Polyoma virus infection causes acute interstitial nephritis and ureteral stenosis in humans but has rarely been noted in other species. In the present study, a hitherto unknown polyoma virus was detected in 12 of 57 cynomolgus monkeys after 3 to 11 weeks of immunosuppression given to promote acceptance of renal allografts or xenografts. This virus, termed cynomolgus polyoma virus (CPV), is antigenically and genomically related to simian virus 40 (SV40). The tubular epithelial nuclei of the collecting ducts in the medulla and cortex reacted with an antibody for the SV40 large T antigen and by electron microscopy contained densely packed paracrystalline arrays of 30- to 32-nm diameter viral particles. A polymerase chain reaction analysis of DNA extracted from affected kidneys detected polyoma virus sequences using primers for a highly conserved region of the large T antigen of polyoma virus. Sequence analysis showed 7 base substitutions and 3 to 5 deletions in the 129-nucleotide segment of amplified products, compared with the corresponding portion of SV40, yielding 84% homology at the amino acid level. CPV caused interstitial nephritis in six renal allografts, a xenograft kidney, and six native kidneys. Infected animals showed renal dysfunction and had tubulointerstitial nephritis with nuclear inclusions, apoptosis, and progressive destruction of collecting ducts. CPV was detected in the urothelium of graft ureters, associated with ureteritis and renal infection. Viral infection was demonstrable in smooth muscle cells of the ureteric wall, which showed apoptosis. One animal had diarrhea and polyoma virus infection in the smooth muscle cells of the muscularis propria of the intestine. Spontaneous resolution occurred in one case; no animal had virus detected in tissues more than 3 months after transplantation. Thus, immunosuppression predisposes cynomolgus monkeys to a polyoma virus infection with clinical consequences quite similar to BK virus infection in humans, including renal dysfunction. We also suggest that this may be the pathogenetic basis for the significant incidence of late onset, isolated ureteral stenosis observed in these recipients.

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.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.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.To our knowledge, this is the first report of long-term survival of discordant xenogeneic BM in a primate recipient.

The renal allograft biopsy is generally accepted as the gold standard for clarifying the cause of renal dysfunction. However, the clinical usefulness of this procedure has rarely been studied prospectively, nor have most studies included follow-up of patients to delineate the influence of the biopsy on clinical outcome. In this study, we evaluated prospectively the clinical usefulness of the allograft biopsy in renal transplant recipients receiving cyclosporine (CyA).During a 21-month period, 82 biopsies were performed. In 54 instances (47 patients), we outlined a presumed diagnosis and tentative treatment plan before the procedure. After the biopsy, a definitive diagnosis was made and an appropriate patient management approach was instituted. We analyzed the incidence of change in patient management that resulted from histological findings. All patients were followed to monitor their response to treatment and allograft survival. In cases of biopsy-proven acute cellular rejection (ACR) or cyclosporine (CyA) toxicity, clinical and laboratory data from the day of the biopsy were reviewed to determine their diagnostic value.One biopsy specimen was inadequate for definitive interpretation. The biopsy findings resulted in a change in patient management in 22 (41.5%) of the remaining 53 cases (change group). The incidence of altered patient management was 38.7% in biopsy specimens taken in the first month, 55.6% between 1 and 12 months, and 38.5% after 1 year posttransplantation. A change in management was required in 2 of 2 patients with chronic allograft dysfunction, in 44.4% of the 45 patients with acute allograft dysfunction, and in none of the patients with delayed graft function (n=6). Within the first week of treatment 19 of 22 (86.4%) in the change group and 25 of 31 (80.6%) in the no change group had a positive response to therapy. The 1-year allograft survival rate was also similar between the two groups. None of the clinical and laboratory data was useful in distinguishing ACR from CyA toxicity.Renal allograft biopsy findings alter patient management recommendations in approximately 40% of patients in whom a presumptive diagnosis had been made on the basis of clinical and laboratory findings. Patients who had a change in patient management because of biopsy findings demonstrated a response to therapy and allograft survival similar to those of patients who had no alteration in management plan after the biopsy.

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.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.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+/-16,388 in A and $193,525+/-14,444 in B (P=0.288). The increased charges were associated with longer inpatient length of stay (LOS) before transplant, resulting in increased pretransplant charges from $24,088+/-4134 (A) to $39,490+/-6,196 (B) (P=0.011). Room and service (54%) was the largest pretransplant contributor to charges, while blood products (23%), room and service (21%), organ acquisition (13%), and operating room charges (11%) contributed the most after transplant.Longer waiting times resulting in transplantation at later stages of disease have occurred, leading to longer pretransplant LOS and its associated charges. Despite more advanced disease, patient survival rates have significantly improved with fewer infection-related deaths. LOS pretransplant, blood products, and operating room services represent potential areas for providing more cost-effective care.

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.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.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 microg/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 (P0.05). As compared with extracorporeal membrane oxygenation, which activated classical and alternative pathways, OKT3 predominantly activated complement by the classical pathway. Methylprednisolone pulses did not activate complement.Complement activation is an early event after OKT3 administration and is associated with the increased expression of adhesion molecules on neutrophils and with pulmonary hemodynamic changes. Effective therapeutic approaches to the control of early monoclonal antibody side effects may require measures that limit complement activation in addition to reducing cytokine activity.

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.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.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.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.

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.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.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.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.

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.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 recipient's serum at the time of rejection. Pretransplant donor-specific T- and B-cell cross-matches were negative.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).Our findings suggest that a therapeutic approach combining PE and tacrolimus-mycophenolate mofetil rescue has the potential to improve the outcome of AHR.

Ongoing studies at our center on facilitating transplantation of discordant xenogeneic organs are focused on tolerance induction. To abrogate hyperacute rejection, we have used adsorption methods to eliminate natural anti-Gal(alpha)1-3Gal (alphaGal) antibodies from the circulation of baboons. We have analyzed data concerning antibody removal in baboons that were 1) immunologically naive, 2) receiving conventional pharmacologic immunosuppressive therapy (IS), and 3) treated with a conditioning regimen for tolerance induction. We compared the efficiency of removing alphaGal antibody 1) by perfusion of whole blood through an alphaGal affinity column (CP; n=5) with 2) perfusion of plasma (separated from cellular components by apheresis) through an alphaGal column (CPA; n=39). Our studies demonstrate that 1) CP and CPA are equally effective in removing anti-alphaGal antibody, 2) CPA is the method of choice if multiple adsorptions are required, 3) CPA in naive animals transiently affects levels of total IgG and IgM, 4) four CPAs repeated at 2-4 day intervals in association with heavy IS reduce the pool of anti-alphaGal antibody and total Ig, and 5) splenectomy and/or IS delay the return of anti-alphaGal antibody.

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-alpha 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.

Collapsing glomerulopathy is a recently described form of glomerular injury characterized by capillary collapse and visceral epithelial hypercellularity associated with nephrotic range proteinuria and a rapid, progressive decline in renal function. The lesion has rarely been described in allografts.We reviewed 892 allograft biopsies from a population of 1079 recipients who received renal transplants between 1978 and 1996.Five cases of de novo collapsing glomerulopathy were identified (0.6% of biopsies; 3.2% since 1993). None occurred before 1993. The patients were 31 to 66 years of age and they presented 6 to 25 months after transplantation. The 24-hr urinary protein ranged from 1.8 to 11.8 g. All patients and donors were negative for the human immunodeficiency virus and had no risk factors for human immunodeficiency virus infection. Diffuse or focal, global or segmental collapse of glomerular capillaries, swelling and hypercellularity of the visceral epithelium, hyaline arteriolosclerosis, and interstitial fibrosis were characteristic histologic features. Two cases had concomitant glomerular immune complex deposits. Progressive decline in allograft function occurred within 2-24 months after diagnosis, culminating in return to dialysis in all patients.Collapsing glomerulopathy can arise in renal allografts as a de novo disease. Although its pathogenesis remains to be clarified, it is important to distinguish this lesion in allografts as it can be associated with rapidly progressive graft failure.

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.Constructs of humanized OKT4A containing the complementarity-determining region (CDR) of murine OKT4A and the framework and constant regions of human light (kappa) 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 mg/kg) or as low-dose multiple infusion (1 mg/kg for 12 days) was given, and the effects on graft survival, immunohistology, circulating cells, and lymph node cells were assessed.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 (23.8+/-13.2 days vs. 7.4+/-1.5 days, P0.001) or interleukin-2-promoted T-cell outgrowth from allograft biopsies (22.2+/-11.0 days vs. 6.3+/-0.5 days, P0.01).This study demonstrates that the in vivo effects of CDR-grafted OKT4A are dependent on its isotype. The depleting mAb CDR-OKT4A/hIgG1 significantly delays the entry of CD4+ cells into the graft, inhibiting the early phase of rejection. However, graft rejection occurs when CD4+ cells eventually infiltrate the graft, even in the presence of depressed levels of circulating CD4+ cells. Both isotypes demonstrated therapeutic efficacy: graft survival was prolonged over controls. In the case of CDR-OKT4A/hIgG4, neither lymphocyte depletion, antigenic modulation, nor prevention of infiltration is necessary for a beneficial effect, which indicates that this mAb blocks CD4 function or renders the CD4+ cell less responsive. The lack of depletion is a feature of potential clinical advantage in minimizing the risk of excessive immunosuppression.

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.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.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 (P0.05), respectively. In both groups, there was a significant correlation between total GFR and allograft GFR (P0.001), 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.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.

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.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.Overall, 22 of 35 (63%) liver recipients had a positive anticardiolipin antibody test (either IgG or IgM titer4 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.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.

We demonstrated previously that a nonmyeloablative preparative regimen can induce mixed chimerism and allograft tolerance in cynomolgus monkeys.The current studies were designed to clarify the importance and toxicity of various elements of the allotolerance conditioning regimen by: fractionating or reducing the whole-body irradiation (WBI) dosage; adding deoxyspergualine; or deleting donor bone marrow, cyclosporine, irradiation, or splenectomy.Monkeys treated without donor bone marrow, cyclosporine, or irradiation did not develop chimerism or long-term allograft survival. One of three monkeys treated without splenectomy developed chimerism but died of a surgical complication. The other two did not develop chimerism and rejected by day 117. Six of six monkeys treated with 300 cGy of fractionated WBI developed chimerism. Five of these recipients had long-term graft survival. Only two of four monkeys treated with 250 cGy developed chimerism, so a 2-week course of deoxyspergualine was added. This led to chimerism in two monkeys, but one died of ureteral stenosis and the other died of allograft rejection. An unanticipated high incidence of ureteral complications felt to be secondary to rejection episodes and ischemic injury was observed in the long-term surviving animals.All parameters of the original preparative regimen seem to be essential for consistent success. The degree of lymphocyte depletion was proportional to the WBI dose. Long-term graft survival was observed only in recipients achieving lymphocyte chimerism of1.5%. In this model, lymphocyte depletion seems to be the best predictor of chimerism, and significant lymphocyte chimerism seems to be important in achieving tolerance.

Melanoma can remain clinically quiescent for decades before regional or distant recurrence appears. This protracted disease free interval challenges the concept of a "cure" for melanoma.To understand this prolonged dormancy better, the authors retrospectively studied patients who developed recurrent melanoma 15 years or longer after their initial diagnosis ("ultra-late" recurrence). These cases were identified from 2766 melanoma diagnoses available in the Cancer Registry at the Massachusetts General Hospital (MGH). Histologic features of the primary lesion were also included when possible.Twenty cases were retrieved from the MGH database. There were equal numbers of women and men, although women were younger at the time of initial diagnosis (mean age of women: 29.8 years vs. 43.0 years for men). No patients had more than one primary cutaneous melanoma. The trunk was the most common primary site (35%), although there was no predominant anatomic localization. The average disease free interval was 17.3 years for women, 20.0 years for men, 18.1 years for patients with regional recurrence, and 19.0 years for patients with distant metastases. Distant recurrence was the most common type of recurrence (50% of women and 60% of men). The estimated probability of survival (5 years after recurrence) was 0.8 for regional disease and 0.2 for distant disease. With the available histologic records, it appears that almost all tumors were Clark Level III or IV with thicknesses ranging from 0.8-2.3 mm. In contrast to the published cases, this study did not find that women with lower extremity melanomas were at higher risk for developing ultra-late recurrence.Ultra-late recurrence of melanoma, although uncommon, can occur in any patient without identifiable risk factors. Because many prognostically favorable melanomas (thin melanomas on extremities) can recur after prolonged disease free intervals, the possibility of delayed recurrence remains and must be kept in mind.

A preliminary clinical study of renal allograft recipients revealed that a dimeric form of the human 80 kDa soluble receptor (sTNFR:Fc) for tumor necrosis factor (TNF) is well tolerated and attenuates the OKT3-induced acute clinical syndrome. The current study determined the in vivo biological effects and fate of sTNFR:Fc in these patients. Serial assessment of both antigenic and biological activities of circulating TNF and sTNFR:Fc have led to the following observations. (1) Although control patients typically responded to the first OKT3 injection with a rapid increase of biologically active TNFalpha, patients on sTNFR:Fc therapy had markedly higher serum TNFalpha antigenic levels, but no detectable bioactivity. Thus, sTNFR:Fc functioned as a potent antagonist, despite its cytokine-carrier effect. (2) Peak sTNFR:Fc levels averaging 800 and 2500 ng/ml were routinely achieved in vivo, using the low-dose (0.05 mg/kg) and high-dose (0.15 mg/kg) protocols. (3) The half-life of circulating sTNFR:Fc was estimated to be approximately 4.4 days, and levels of p80 receptors in treated patients remained significantly above those in control patients for at least 20 days. (4) In vitro blocking studies demonstrated that circulating sTNFR:Fc remained biologically active for 2 weeks. These results demonstrate that under current protocols, significant serum levels of sTNFR:Fc, capable of effectively neutralizing TNF activity over prolonged periods, can be achieved. The persistent OKT3 side effects observed, despite sTNFR:Fc therapy, are therefore likely to be caused by factors other than TNF.

Because they can be selected to target only cells which are crucial for rejection, monoclonal antibodies (mAbs) offer enormous potential for specific manipulation of the immune response. Interest in the clinical potential of anti-CD4 mAbs has been heightened by the demonstration, in experimental models, that such therapy can produce long-term donor-specific non-responsiveness. Early clinical trials using two murine anti-CD4 mAbs (BL4 and mT-151) were discouraging, with over 50% of recipients suffering early rejection episodes. Another murine preparation, OKT4A, was initially found to prolong allograft survival in non-human primates. Limited clinical trials revealed that this mAb was well tolerated, that most recipients produced an antimurine response, and that only 26% of patients suffered rejection episodes during the first 3 post-operative months. Another murine preparation, Max. 16H5, has been reported to reverse late onset acute rejection episodes as effectively as, but more safely than, conventional immunosuppression. More recent interest has focused upon humanized recombinants of these earlier murine anti-CD4 preparations. cMT-412, has been studied in recipients of heart or heart-lung allografts. These patients were observed to have less frequent and markedly delayed rejection episodes, fewer infectious complications, and better overall survival than that observed in an ATG-treated control group. Further studies are thus being undertaken. A CDR grafted IgG4 preparation of OKT4A has also been studied. This molecule (OKTcdr4a) contains only 8% of the parent murine sequence while retaining the binding affinity of OKT4A for the human CD4 antigen. In a pilot trial, biopsy-proven reversible rejection episodes were observed in 2/11 (18%) of renal allograft recipients. There were no allograft failures and no antibody response to the mAb. These and other trials emphasize the intense interest in immunosuppressive regimens incorporating anti-CD4 mAbs as well as the difficulties encountered in defining optimal protocols. Nevertheless, the impressive results observed in rodent and non-human primate models suggest that these agents are likely to play an important role in future immunosuppressive protocols, particularly those designed to induce tolerance.

The use of veno-venous bypass (VVB) during the anhepatic phase of orthotopic liver transplantation (OLT) remains controversial. We employ VVB on a selective basis: patients who tolerate intra-operative supra-hepatic IVC test cross-clamping undergo OLT without VVB while patients who, despite maximal volume resuscitation, develop hemodynamic instability during test cross-clamping, undergo OLT with VVB. The records of 150 adult orthotopic liver allograft recipients transplanted at the Massachusetts General Hospital from January 1984 to December 1994 were reviewed to identify any potential adverse affects on peri-operative, 6 months, 1 year outcomes in recipients foregoing VVB during liver transplantation. Thirty-eight patients (25%) underwent OLT without VVB with actuarial survivals of 78.4% and 69% at 6 months and 1 year. 112 patients (75%) underwent OLT with VVB with actuarial survivals at 6 months and 1 year of 73% and 72%. Demographic data, UNOS status, and diagnoses were similar in each group. There were no significant differences in intra-operative PRBC requirements; lengths of hospital stay; retransplantation rates; or 30 day, 6 months and 1 year survivals between these two groups. There was no significant difference in renal function as determined by preoperative, peak post-operative, discharge serum creatinine levels, or number of patients requiring HD between these two groups. There were two major complications (1.8%) possibly resulting from VVB. In conclusion, patients who tolerate IVC test cross-clamping can safely undergo orthotopic liver transplantation without veno-venous bypass. In our experience, there were no significant differences in peri-operative parameters, post-operative renal function, or short-term survival when compared to patients who, due to hemodynamic instability during IVC cross-clamping, underwent OLT with VVB. Given the potential complications associated with VVB, we feel that in those patients who tolerate intra-operative IVC cross-clamping, it is better to proceed without the use of VVB.

Liver transplantation for patients requiring life-support results in the lowest survival and highest costs. A ten year (1983-1993) regional experience with liver transplantation for critically ill patients was undertaken to ascertain the fate of several subgroups of patients. Of the 828 liver transplants performed at six transplant centers within the region over this period, 168 (20%) were done in patients who met today's criteria for a United Network of Organ Sharing (UNOS) status 1 (emergency) liver transplant candidate. Recipients were classified according to chronicity of disease and transplant number (primary-acute, primary-chronic, reTx-acute, reTx-chronic). Overall one-year survival was 50% for all status 1 recipients. The primary-acute subgroup (n = 63) experienced a 57% one-year survival compared with 50% for the primary-chronic (n = 51) subgroup (P = 0.07). Of the reTx-acute recipients (n = 43), 44% were alive at one year in comparison with 20% for the reTx-chronic (n = 11) group (P = 0.18). There was no significant difference in survival for the following: transplant center, blood group compatibility with donors, age, preservation solution, or graft size. For patients retransplanted for acute reasons (primary graft nonfunction (PGNF) or hepatic artery thrombosis [HAT]), survival was significantly better if a second donor was found within 3 days of relisting (52% vs. 20%; P = 0.012). Over the study period progressively fewer donor organs came from outside the region. No strong survival-based argument can be made for separating, in allocation priority, acute and chronic disease patients facing the first transplant as a status 1 recipient. Clearly patients suffering from PGNF or HAT do far better if retransplanted within 3 days. Establishing an even higher status for recipients with PGNF, perhaps drawing from a supraregional donor pool, would allow surgeons to accept more marginal donors, thus potentially expanding the pool, without significantly compromising patient survival. Retransplantation of the recipient with a chronically failing graft who deteriorates to the point of needing life-support is nearly futile, and in today's health care climate, not an optimal use of scarce donor livers.

Actinomycin D is a potent cytotoxic agent which inhibits DNA transcription by DNA-dependent RNA polymerases and has previously been used as adjunctive immunosuppressive agent for refractory renal allograft rejection.To assess the efficacy of actinomycin D in cardiac allograft rejection, we studied seven patients with recurrent cellular or humoral rejection. All patients received intravenous actinomycin D 5 micrograms/kg every 6 weeks.During the 6-months after initiation of actinomycin D treatment, the mean number of treated cellular or humoral rejection episodes per patient (0.14) and the mean number of International Society for Heart and Lung Transplantation grade 2 or higher endomyocardial biopsy specimens per patient (0.3) were lower compared with those observed during the 6-month pre-actinomycin D period (1.6 and 2.6, respectively). By 6 months after initiation of actinomycin D, all seven patients were receiving lower daily maintenance doses of corticosteroids. The mean total corticosteroid dose after actinomycin D administration per patient per month (615 +/- 177 mg) was significantly lower than the pre-actinomycin D dose (1012 +/- 347 mg; p = 0.019). No patient had significant adverse effects.Actinomycin D is an effective immunosuppressive agent for prevention of recurrent cellular or humoral rejection after cardiac allograft rejection. The corticosteroid sparing effect of actinomycin D may be of particular benefit.

Tumor necrosis factor alpha (TNFa) and lymphotoxin (LT) or TNF beta are closely linked cytokines produced by macrophages and activated T lymphocytes, which play important regulatory roles in the immune response to allografts. They have also been implicated as mediators of the adverse reactions observed during OKT3 therapy. Therefore, anti-TNF agents could be useful both for immunosuppression and for limiting the systemic response observed in patients receiving OKT3. Recombinant TNFR:Fc is a fusion protein that binds TNFa and LT, thereby neutralizing their effects in vitro. The present study investigates the potential clinical application of TNFR:Fc in a nonhuman primate renal allograft model. Cynomolgus renal allograft recipients were treated with TNFR:Fc induction therapy alone or in combination with subtherapeutic doses of cyclosporine. Control animals received no immunosuppression or subtherapeutic cyclosporine. TNFR:Fc, administered as the only immunosuppressive agent, successfully prolonged renal allograft survival in the majority of treated animals. The prolongation of allograft survival was even more impressive when TNFR:Fc was combined with subtherapeutic doses of cyclosporine. Onset of rejection was significantly delayed as well in the TNFR:Fc treated groups. No adverse side effects were observed in any of the TNFR:Fc treated animals. Precursor cytotoxic T cells were detected in peripheral blood samples of treated recipients but the level of effector CTLs in vivo was below the threshold of detection. These results demonstrate that TNFR:Fc can be safely administered and is effective in prolonging renal allograft survival and in delaying the onset of rejection when administered alone or in combination with cyclosporine.