Improved flow cytometric detection of HLA alloantibodies using pronase: potential implications in renal transplantation.

BACKGROUND: Flow cytomeric crossmatch (FCXM) has grown in popularity and has become the "standard of practice" in many programs. Although FCXM is the most sensitive method for detecting alloantibody, the B cell FCXM has been problematic. Difficulties with the B cell FCXMs have been centered around high nonspecific fluorescence background owing to Fc-receptors present on the B cells and autoantibodies. To improve the specificity and sensitivity of the B cell FCXM, we utilized the proteolytic enzyme pronase to remove Fc receptors from lymphocytes before their use in FCXM. METHODS: Lymphocytes isolated from peripheral blood, spleen, or lymph nodes were treated with pronase and then used in a three-color FCXM. A total of 167 T- and B cell FCXMs using pronase-treated and untreated cells were performed. Testing used serial dilutions of HLA allosera (22 class I and 6 class II), with the titer of each antibody at one dilution past the titer at which the complement-mediated cytotoxicity anti-human globulin crossmatch became negative. RESULTS: After pronase treatment, the actual channel values of the negative control in both T cell and B cell FCXMs declined from 78+/-10 to 57+/-4 (P<0.05) and 107+/-11 to 49+/-3 (P<0.00001), respectively. Pronase treatment resulted in improved sensitivity of the T and B cell FCXM in detecting class I antibody by 20% and 80%, respectively. In no instance was a false-positive reaction observed. In this study, pronase treatment improved the specificity of B cell FCXM for detecting class II antibodies from 75% to 100% (P=0.03). In no instance was a false-negative reaction recorded. Lastly, on the basis of these observations we re-evaluated three primary transplant recipients who lost their allografts because of accelerated rejection. One of the patients was transplanted across negative T and B cell FCXM, whereas the other two patients were transplanted across a positive T cell, but negative B cell, FCXM. After pronase treatment, T and B cell FCXMs of each patient became strongly positive, and donor-specific anti-HLA class I antibody was identi. fied in each case. CONCLUSION: Utilization of pronase-treated lymphocytes improves both the sensitivity and specificity of the FCXM.

Frequency, potential risk and therapeutic intervention in end-stage renal disease patients with antiphospholipid antibody syndrome: a multicenter study.

BACKGROUND: Antiphospholipid antibody syndrome (APAS) is characterized by the presence of anticardiolipin antibodies (ACA) in association with thrombotic disorders of arterial and/or venus systems, spontaneous abortion(s) or thrombocytopenia. METHODS: In this multicenter study, 502 end-stage renal disease (ESRD) patients awaiting renal transplants were screened to determine the frequency of APAS, the potential risk associated with APAS, and strategies for therapeutic intervention. Ninety-three patients (19%) had high titers of ACA. Twenty-three patients had documented evidence of one or more of the thrombotic disorders such as lupus, frequent abortions, frequent thrombosis of arteriovenous shunts, biopsy-proven microrenal angiopathy, or thrombocytopenia and thus were diagnosed with APAS. Of these 23 patients, 11 received kidney transplants either with (4 patients) or without (7 patients), concomitant anticoagulation therapy. RESULTS: All seven of the patients with APAS not treated with anticoagulation therapy lost their allografts within 1 week as a result of renal thrombosis. In contrast, three out of four transplant patients with APAS treated with anticoagulation therapy maintained their allografts for over 2 years. The fourth patient lost his graft within a week because of thrombosis. Of the remaining 70 patients with high titers of ACA but no evidence of thrombotic disorders, 37 received kidney transplants. None lost their allografts as a result of thrombosis. Our data suggest that, although 19% of our ESRD patients exhibit high titer of ACA, only 5% of the patients have APAS. CONCLUSION: In conclusion, our data suggest that the patients with APAS are at high risk of posttransplant renal thrombosis. Anticoagulation therapy could prevent patients from posttransplant thrombosis in patients with APAS.

Hydrocortisone activation of human herpesvirus 8 viral DNA replication and gene expression in vitro.

BACKGROUND: Patients undergoing chronic steroid therapy for organ transplantation are at increased risk for development of human herpes virus 8(HHV-8)-associated Kaposi's sarcoma (KS). It has also been reported that following steroid withdrawal, KS lesions often undergo partial or complete regression. METHODS: We have examined the effect of corticosteroid treatment on HHV-8 replication, gene expression, and lytic protein expression in BCBL-1 cells in vitro. BCBL-1 cells were collected after culture for 24-72 hr with hydrocortisone (HC) 1-5 microM, phorbol ester 20 ng/ml (positive control), and culture medium only (negative control). HHV-8 genomic conformation was examined by Gardella gel analysis. mRNA expression of viral cyclin (v-Cyc), viral Bcl-2 (v-Bcl-2), viral macrophage inflammatory protein-I (v-MIP-I), viral interferon regulatory factor-1(v-IRF-1), and viral tegument protein (TP) was examined by RT-PCR Southern blot. Viral protein expression within the cells was examined by indirect immunofluorescence using 5 different HHV-8 positive antisera from 4 renal transplant recipients and 1 patient with classic KS. RESULTS: Gardella gel analysis revealed that HC induced an accumulation of the linear replicative genomic form of the virus in a time-dependent fashion. Southern blot analysis of the RT-PCR products revealed that HC induced increased expression of v-IRF-1, v-Bcl-2, and TP mRNA, with little discernible effect on v-Cyc, and v-MIP-I. Immunofluorescence revealed that HC induced increased numbers of cells expressing lytic antigens. CONCLUSIONS: These data indicate that hydrocortisone acts directly on BCBL-1 cells to activate the lytic cycle of HHV-8 and provide further support for the hypothesis that HHV-8 is activated in corticosteroid-treated immunocompromised patients.

Serologic and molecular evidence of human herpesvirus 8 activation in renal transplant recipients.

This study was designed to determine whether there is serologic or molecular evidence of human herpesvirus 8 (HHV-8) activation in renal transplant patients, an immunocompromised population at risk for development of Kaposi's sarcoma. Indirect immunofluorescence for detection of HHV-8 serum antibody and Southern blot polymerase chain reaction (PCR) for detection of viral DNA in whole blood were used. Seroprevalence and geometric mean titer (GMT) were significantly increased in the transplant group compared with healthy adults and were comparable to those in human immunodeficiency virus (HIV)-positive adults (transplant patients, 50% [GMT 1:210]; healthy adults, 7% [GMT 1:44]; HIV-positive patients, 73% [GMT 1:172]). Viral DNA was present in the blood of some renal transplant patients (3/33 PCR-positive) but in none of 20 healthy adults. Thus, there is both serologic and molecular evidence of HHV-8 activation in the renal transplant population compared with healthy adults (P<.01). The serologic results approximate those obtained for HIV-positive adults.

Relative risk of post-transplant renal thrombosis in patients with antiphospholipid antibodies.

INTRODUCTION: Antiphospholipid antibody syndrome (APAS) is a condition associated with recurrent arterial and venous thrombosis, recurrent abortions, and thrombocytopenia either with or without lupus. In this study we have evaluated the impact of APAS on the renal transplant outcome of 174 patients. METHOD: Patients' APAS status was determined by the presence of anticardiolipin antibodies (ACA) and a history of clotting disorders. Serum samples from each patient were tested for the presence of ACA by the ELISA method. Transplant outcomes were monitored for > or = 1 yr. RESULTS: Of 174 patients, 78 received renal transplants. Six of these 78 patients had APAS as evidenced by either recurrent microrenal angiopathy (2 patients), thrombocytopenia (1 patient) or frequent A-V shunt thrombosis (3 patients) along with high titers of ACA of IgM, IgG, or both subtypes at the time of their transplants. Each of these 6 patients thrombosed their renal allografts within a week of their transplants. The other 72 transplanted patients with no APAS were all doing well 1 yr post-transplant. The association between APAS and post-transplant renal thrombosis among these patients is highly significant (p < 0.0001). In contrast, no association was discerned between post-transplant thrombosis and prior sensitization to HLA CONCLUSION: Our data demonstrates that patients with APAS are at high risk for development of post-transplant renal thrombosis.

Prevention of graft-versus-host disease by intrathymic injection of recipient-type splenocytes into donor.

We have prevented graft-versus-host disease (GVHD) by tolerizing graft donors to host antigens by intrathymic injection of recipient-type splenocytes into donors. A unidirectional GVHD model was used in which intravenous injection of 3-4 x 10(8) Lewis rat (donor) lymphocytes into (Lewis x Brown Norway)F1 rats (recipients) causes lethal GVHD. The donor animals were divided into five treatment groups. The group 1 donor animals received no treatment. The group 2 donors received a single intraperitoneal injection of 1 ml of antilymphocyte antiserum (ALS). The group 3 donors received an intrathymic injection of 50x10(6) host splenocytes. The group 4 donors received both ALS (intraperitoneally) and intrathymic allograft. The group 5 donors received both ALS (intraperitoneally) and intravenous allograft. Two weeks after these treatments, 3-4x10(8) lymphocytes from each of these donors were injected (intravenously) into the recipients. The clinical signs of GVHD, as measured by profound weight loss, hair loss, inflammation of foot pads and ears, and profound splenomegaly, were evident in recipients of groups 1, 2, and 3 between days 9 and 10 and in the recipients (two of four) of group 5 on day 17. No GVHD was observed by histopathology in all 14 hosts that received lymphocyte injection from the group 4 donor animals (up to 300 days). These results demonstrate that GVHD can be eliminated by tolerizing donors toward host by intrathymic injection of the recipient-type lymphocytes into the donor. A single injection of ALS is necessary to possibly eliminate antihost response from the donor for the tolerance induction. The thymic route appears to be superior to the intravenous route for tolerance induction.

Renal allograft failure after simultaneous pancreas-kidney transplantation: univariate and multivariate analyses of donor and recipient risk factors.

Although donor and recipient risk factors for renal allograft failure are well known after kidney transplantation, they are less well defined after simultaneous pancreas-kidney transplantation. The purpose of this study is to evaluate the impact of donor and recipient risk factors on the outcome of the renal allograft in simultaneous pancreas-kidney recipients. Simultaneous pancreas-kidney transplant performed between 4/88 and 6/94 were reviewed (n = 61) and univariate (Kaplan-Meier) and multivariate (Cox regression) analyses of factors which affect kidney graft survival were performed. Twelve donor and eleven pre- and post-transplant recipient risk factors were evaluated. Overall kidney allograft survival rates at 1, 2 and 5 yr were 81%, 76% and 66%. Donor age > and = 40 yr (RR = 2.3), donor female gender (RR = 3.5), donor admission to pronouncement of brain death > and = 48 h (RR = 3), the occurrence of surgical complications (RR = 2.1), and serum > and = 2 mg/dl on post-transplant day (RR = 1.9) were independently associated with an increased hazard of graft failure. With the exception of length of donor admission, all of these factors were also shown to predict the risk of renal graft failure by univariate analysis. In conclusion, we have identified donor and recipient risk factors which independently predict the risk of renal graft failure after simultaneous pancreas-kidney transplantation. Whether the differences between our center-specific risk factors and those obtained from renal transplant registry data are true differences or simply reflect sampling error is unclear.

Renal retransplants: effect of primary allograft nephrectomy on early function, acute rejection and outcome.

Although risk factors for failure of renal retransplants have been well studied, the impact of allograft nephrectomy on subsequent renal transplantation in the cyclosporin era is not well defined. The purpose of this study is to define the effect of nephrectomy of the primary allograft on subsequent allograft survival, early allograft function, incidence of acute rejection and patient sensitization. The records of 127 renal retransplant recipients were reviewed. Of these 127 patients who underwent retransplantation, 40 (31%) underwent nephrectomy of the primary allograft prior to retransplantation whereas 40 (31%) did not. Nephrectomy of cadaveric primary allografts was performed more commonly (48% vs 30%, p = 0.003) and earlier (78% vs 54% < 1 month post-transplant, p = 0.0006) in the pre-CSA period compared to the CSA period. Biopsy-proven acute rejection episodes occurred more frequently in the nephrectomy group (73% vs 42%, p = 0.03). Although primary allograft nephrectomy was associated with higher preformed antibody levels, it had no effect on early graft function, frequency of acute rejection or allograft outcome after retransplantation, in the CSA group. In conclusion, in the cyclosporin era, nephrectomy of the primary allograft has no significant influence on retransplantation.

Renal allograft and patient outcome after transplantation: pancreas-kidney versus kidney-alone transplants in type 1 diabetic patients versus kidney-alone transplants in nondiabetic patients.

Despite recent advances and improved outcome, pancreas transplantation remains controversial. The purpose of this review was to study renal allograft outcome after simultaneous pancreas-kidney transplants (SPK, n = 61), kidney-alone transplants in type I diabetic patients (KA-D, n = 63), and kidney-alone transplants in nondiabetic patients (KA-ND, n = 80). Patients were matched for donor age, donor gender, donor race, interval from donor admission to procurement, DR mismatch, and recipient gender. The mean renal allograft cold ischemic time and recipient age were lower in the SPK group. Patient survival was highest in the KA-ND group (99% and 86% at 1 and 5 years, respectively), intermediate in the SPK group (90% and 78% at 1 and 5 years, respectively), and lowest in the KA-D group (89% and 66% at 1 and 5 years, respectively) (P = 0.004). similarly, renal allograft survival was higher in the KA-ND (89% and 63% at 1 and 5 years, respectively) and SPK (82% and 69% at 1 and 5 years, respectively) groups compared with the KA-D group (76% and 49% at 1 and 5 years, respectively) (P = 0.07). This difference disappeared when renal graft survival was censored for death, which probably reflects the selection bias. Actuarial pancreas graft survival was 76% and 62% at 1 and 5 years, respectively. Acute rejection (AR) was more frequent in the SPK group than in the KA-D and KA-ND groups (41% v 16% v 29%; P = 0.007). Delayed graft function (DGF), on the other hand, occurred more frequently in the KA-D group than in the KA-ND and SPK groups (66% v 55% v 38%; P = 0.08). Death as a result of a cardiovascular event occurred more frequently in the KA-D group. Cardiovascular death and renal graft failure occurred earlier in the SPK group. Cox regression analysis revealed a 1.6 and 1.8 times higher risk of renal graft failure in the SPK group when the donor was > or = 40 years old or female and a five times higher risk of graft failure in the KA-ND group in the presence of AR. Graft survival in patients with AR/DGF was lower than that in patients with no AR/no DGF in both the KA-D (71% and 63% v 100% and 100% at 1 and 5 years, respectively; P = 0.03) and KA-ND (90% and 56% v 100% and 100% at 1 and 5 years, respectively; P = 0.001) groups. Acute rejection did not affect graft survival in the SPK group. In the absence of AR, DGF had no effect on graft survival in any of the groups. Although the selection bias in favor of pancreas transplantation does not allow for definitive conclusions, our results show that outcome after SPK transplantation is acceptable and factors that influence the outcome after this procedure may be different from the ones affecting KA-D recipients.

Urologic complications after simultaneous pancreas-kidney transplantation: hand-sewn versus stapled duodenocystostomy.

Pancreas transplantation with bladder drainage of exocrine secretions may be associated with significant urologic complications. Stapled and hand-sewn duodenocystostomies were compared in 61 recipients of simultaneous pancreas-kidney transplants. Both methods resulted in similar urologic complication and allograft survival rates. Duodenal segment leaks were associated with significant morbidity and decreased patient and allograft survival.

Multivariate analysis of donor risk factors for pancreas allograft failure after simultaneous pancreas-kidney transplantation.

BACKGROUND: Donor and recipient selection criteria for pancreas allograft are not standardized and may vary from center to center. METHODS: Simultaneous pancreas-kidney transplantations performed between April 1988 and June 1994 were reviewed (n = 61), and univariate and multivariate analyses of factors that affect pancreas graft survival were performed. Analysis of all cases and cases excluding early thrombosis were performed separately. RESULTS: Pancreas graft survival when early thrombosis was excluded and in the overall group was 76% and 70%, respectively, at 1 year. Although blood group and donor gender were weak predictors of graft survival by univariate analysis, neither affected graft survival in the multivariate model. Risk factors for graft failure as determined by Cox regression analysis and in descending order of significance were (1) duration of brain death before procurement, (2) length of donor admission, and (3) donor age of 40 years or older. The risk of graft failure for each of these factors was increased 2.2-, 3.2-, and 4-fold, respectively. Prolonged brain death was the only risk factor in the overall group, suggesting an association with early thrombosis. CONCLUSIONS: Center-specific donor risk factors for pancreas graft survival after simultaneous pancreas-kidney transplantation were identified in this study, the importance of which need to be better defined.

Clinical importance of pre-morteum blood lymphocytes in cadaver donor tissue typing.

We have refined our immunomagnetic bead (IM bead) procedures to isolate pure and viable lymphocyte subpopulation from pre-morteum (PM) blood for cadaver donor HLA typing, preliminary and final crossmatches (XMs). The results of 1220 XMs were compared using T/B lymphocytes isolated either from PM blood or spleen/lymphnode (SPLN) tissue. IM bead technique was used to isolate T/B cells from PM blood and nylon wool column (NWC) technique was used to isolate T/B cells from SPLN. When we compared the outcome of 800 T-cell crossmatches using T cells from PM blood or SPLN of 5 separate cadaver donors, NWC TXMs tended to be more falsenegative for high PRA (> 10%, total 500 XMs) as well as low PRA (< 10%, total 300 XMs) did not reach statistical significance. In contrast, NW BXM (420 B XM) were found to be far more false negative than IM bead BXM regardless of the PRA of the patients. In order to ensure that NWC BXMs were indeed false negative, 23 sera with known anti-DR antibodies were BXMed where antigen-specific B cells were isolated by both the techniques. Our results showed that IM bead BXM identified the DR specificities greater than 90% of the time, the titers of ab specificities were stronger (1:8). In comparison, NWB cell XMs were weak (titers 1:2), and the false negative rate for some ab was as high as 73%. Using IM bead and NWC techniques we compared our turnaround time (TAT) for cadaver donor typing, preliminary and final XMs.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of FK506 and cyclosporine on dynamic insulin secretion from isolated dog pancreatic islets.

Pancreatic islet transplantation may be the most ideal treatment for patients with insulin-dependent diabetes mellitus. However, immunosuppressive agents such as cyclosporine A(CsA) and FK506, used for these transplanted patients have been reported to cause glucose intolerance. In the present study, we have compared the effects of CsA and FK506 on glucose-stimulated insulin release from the isolated dog pancreatic islets, which have been maintained in culture for 3 days after isolation. The isolated dog pancreatic islets, pretreated for 24 hr with either CsA or FK506 (1, 10, and 100 nM), were perifused with 16.7 mM glucose. Pretreatment with both drugs suppressed glucose-stimulated insulin secretion in a dose-dependent fashion. CsA (100 nM), which is a therapeutically relevant concentration, significantly suppressed both the first and second phases of glucose-stimulated insulin release compared with 100 nM FK506. These findings suggest that, with a therapeutically relevant concentration, FK506 may be less toxic than CsA against pancreatic islets in patients with organ or cell transplantation.

Cystadenoma and cystadenocarcinoma with mesenchymal stroma of the liver. Immunohistochemical analysis.

Cystadenoma with mesenchymal stroma is a rare neoplasm of the liver that occurs exclusively in young women and has a potential for malignant transformation. A light microscopic and immunohistochemical study of a case of biliary cystadenoma and another of biliary cystadenocarcinoma revealed a range of differentiation of the lining epithelial cells. The lining cells in the cystadenoma resembled the cells of the normal intrahepatic bile ducts. In contrast, the epithelial lining in the case of cystadenocarcinoma had features of intestinal mucosa, including goblet, Paneth, and endocrine cells similar to those found in other mucinous cystic neoplasms of the foregut area. The compact "ovarianlike" mesenchymal stromal cells had immunohistochemical characteristics of myofibroblasts. These are reactive contractile cells that may proliferate in response to the expanding cysts and female hormones, and they differ immunohistochemically from ovarian stromal cells.

Pancreas transplantation. A new program.

Sixteen pancreatico-duodenal transplants were performed on 15 insulin-dependent diabetics, aged 25-46, during a 20-month period beginning May 1, 1988. Fourteen patients received a combined cadaveric pancreas/renal transplant with bladder drainage. One patient received a second pancreas transplant 24 hours after the first pancreas graft failed due to portal vein thrombosis. One patient received a pancreas graft 3 years after kidney transplantation. Complications included five cases of hematuria, two bladder leaks, two wound infections, one cytomegalovirus pneumonia, three cases of graft pancreatitis, one pseudocyst, one urine reflux pancreatitis requiring conversion to pancreatico-enterostomy, and two late deaths. Average time to discharge was 17 days following transplant, with 2.9 re-hospitalizations per patient and an average of 38 in-hospital days during the first 6-12 months. Seventeen rejection episodes occurred in 12 patients, diagnosed by declining urine amylase and pH and/or finding of rejection on kidney biopsy. Patient and kidney graft survival is 87 per cent. Pancreas graft survival is 81 per cent (1-20 months follow-up). All patients are insulin-independent and normoglycemic. Mean glycosylated hemoglobin concentration is 4.0 +/- 0.9 post-transplant vs. 7.5 +/- 0.6 pretransplant. Mean serum creatinine is 1.4 +/- 0.7 mg/dl. A new program of pancreas transplantation can be successful in carefully selected diabetic patients, with special attention to avoidance of preservation injury to the pancreas during multiorgan donor procurement. Combined pancreatic/renal transplantation is believed to be the therapeutic treatment of choice in Type I diabetic patients who have impaired renal function and have no significant cardiovascular disease.