Development and application of an enzyme-linked immunosorbent assay for the quantitation of alternative complement pathway activation in human serum.

We have developed a new, specific, and highly sensitive enzyme-linked immunosorbent assay (ELISA) which quantitates activation of the alternative pathway in human serum, plasma, or on the surface of activators. The ELISA detects the third component of complement (C3b), proteolytic fragment of complement Factor B (Bb), and properdin (P) complex or its derivative product, C3b,P. In the method, activator-plasma mixtures, plasma containing an activated alternative pathway, or other samples are added to the wells of microtitration plates precoated with antibody to P. C3b, Bb,P or C3b,P complexes which become bound are quantitated by subsequently added, enzyme-labeled, anti-C3. The resulting hydrolysis of the chromogenic substrate is expressed as nanograms of C3b by reference to a C3 standard curve. In addition to absolute specificity for activation of the pathway because of the nature of the complex detected by the assay, the ELISA is highly sensitive and able to reproducibly detect 10-20 ng/ml of C3b,P complexes in serum. This value corresponds to 0.0015% of the C3 in serum. In a series of studies to validate the parameters of the ELISA, reactivity was found to be dependent on the presence of alternative pathway proteins, the functional integrity of the pathway, and on the presence of magnesium. Sheep erythrocytes were converted to activators by treatment with neuraminidase. By using a variety of activators, the kinetics of activation and the numbers of bound C3b molecules quantitated by the ELISA were very similar to those measured by C3b deposition. The ELISA also detected identical activation kinetics when MgEGTA-serum and a mixture of the purified alternative pathway proteins were used as sources of the pathway. ELISA reaction kinetics also correlated with the restriction index, a measure of alternative pathway-activating ability. These studies cumulatively validate the ELISA as a direct and quantitative assay for alternative pathway activation. The sensitivity of the ELISA has permitted its use to detect direct alternative pathway activation by several viruses. The ELISA has also shown that certain classical pathway activators trigger the amplification loop of the alternative pathway while others do not. In addition, stable ELISA reactive complexes appeared in the supernatant of mixtures of serum with certain, but not other activators. The ability of the ELISA to detect activation which has already occurred and the stability of the reactive complexes permits studies of clinical sera. Normal human sera (20) contained low levels (5-20 ng/ml) of ELISA-reactive complexes. A proportion of sera from individuals with the adult respiratory distress syndrome (9-10), typhoid fever (8-10), malaria (3-5), gram-negative sepsis (9 of 47), acute trauma and shock (6 f 25), and systemic lupus erythematosus (3 of 29) showed elevated levels of complexes reactive in the alternative pathway ELISA. In contrast, nine sera from patients with circulating C3 nephritic factor were not reactive in the ELISA.

Complement activation by the surface of Plasmodium falciparum infected erythrocytes.

The surface of trophozoite-stage Plasmodium falciparum infected erythrocytes will, in the presence of immune human or owl monkey serum, activate the classical complement pathway. This was demonstrated with a sensitive, enzyme-linked immunosorbent assay which detects the complex, C1s-C1 inhibitor, which is only generated when the classical pathway is activated. A second enzyme-linked immunosorbent assay, as well as Covaspheres coated with affinity-purified anti-C3, showed that immune activation of the classical pathway by infected erythrocytes resulted in the accumulation of significant amounts of C3b on the erythrocyte surface. During the development of the parasite to the trophozoite stage, the erythrocyte membrane is also transformed from a non-activator into a surface capable of activating complement by the alternative pathway. Erythrocytes infected with trophozoite-stage parasites directly activated the alternative complement pathway. This activation led to the specific binding of an average of 15,000 C3b molecules per infected cell. Alternative pathway activation was augmented by anti-parasite antibody. Such conditions mediated the accumulation of an average of 36,000 C3b molecules per infected erythrocyte. The amounts of C3b on the infected erythrocyte surface did not lead to cellular lysis. They are, however, likely to have a major impact on the total in vivo response to this parasite.

Inhibition of anti-OKT3 antibody generation by cyclosporine--results of a prospective randomized trial.

To investigate the influence of therapy with cyclosporine on the generation of antibodies to OKT3, 51 renal transplant recipients previously maintained on CsA, azathioprine, and prednisone were allocated randomly either to receive 50% of their maintenance dose of CsA (group 1, n = 27) or to discontinue CsA (group 2, n = 24) during treatment with OKT3 for acute renal allograft rejection. In the month following therapy with OKT3, anti-OKT3 antibodies were detected in 11% of patients in group 1 and in 42% of patients in group 2 (P less than 0.02). No patient in group 1 developed antibody titers greater than 1:100, whereas 4 patients in group 2 developed titer greater than or equal to 1:1000. Rejection was reversed in 96% of patients in group 1 and in only 75% of patients in group 2 (P less than 0.03), suggesting that continued administration of reduced doses of CsA during therapy with OKT3 improves the short-term response to this monoclonal antibody. Results of this study suggest that concurrent administration of CsA during therapy with OKT3 inhibits the generation of anti-OKT3 antibodies and improves the response to this monoclonal antibody.

Reexposure to OKT3 in renal allograft recipients.

Between 40% and 80% of patients treated with the monoclonal antibody OKT3 develop blocking antibody against its idiotypic region. Thus a major concern with the use of OKT3 as part of a baseline immunosuppressive regimen is that formation of blocking antibodies might preclude its subsequent use. Between 7/86 and 2/87, 32 patients received prophylactic OKT3 in addition to low-dose prednisone, azathioprine, and cyclosporine. Prophylactic OKT3 did not prevent rejection, as 21 of 32 patients studied developed rejection. Retreatment of 13 patients with OKT3 successfully reversed 12 rejections and lowered the number of T3-positive cells in spite of a low level of blocking antibody in two patients in this group. Of the patients analyzed, 38% developed blocking antibody on initial exposure to OKT3, but OKT3 reuse was denied only 4 patients due to the presence of these antibodies. Three of these had rejections reversed with steroids alone; the other patient lost the allograft. A high frequency of infectious complications occurred in the retreatment group, with viral infections predominating. Only one patient in the retreated group developed antibodies after the second use. Appearance of blocking antibodies after use of OKT3 as part of a base-line prophylactic immunosuppressive regimen did not significantly compromise access to OKT3 for treatment of subsequent rejection episodes, but multiple exposures to OKT3 did increase the frequency of infectious complications.

The effects of steroid withdrawal on the lipoprotein profiles of cyclosporine-treated kidney and kidney-pancreas transplant recipients.

Lipoprotein profiles were measured before and two months after complete withdrawal of prednisone in 34 kidney and 9 kidney-pancreas transplant recipients subsequently maintained on cyclosporine and azathioprine. Withdrawal of steroid therapy was accompanied by a 17% reduction in total serum cholesterol levels. However, there was a parallel reduction in all other measured lipoprotein concentrations, including an 18% reduction in high-density lipoprotein cholesterol levels. In diabetic recipients of a kidney or kidney-pancreas transplant, the ratio of total to high-density lipoprotein cholesterol was unchanged after steroid withdrawal. In nondiabetic kidney transplant recipients, this ratio actually increased significantly following withdrawal of prednisone. These observations suggest that it is premature to presume that withdrawal of steroid therapy will reduce the cardiovascular risk related to hyperlipidemia in cyclosporine-treated kidney or kidney-pancreas transplant recipients.

A prospective randomized trial of prednisone versus no prednisone maintenance therapy in cyclosporine-treated and azathioprine-treated renal transplant patients.

The purpose of this study was to evaluate early (6-12 days) prednisone withdrawal in cyclosporine- and azathioprine-treated renal transplant recipients. Patients, including 8 recipients of live-related donor kidneys and 59 recipients of cadaver donor kidneys, were prospectively randomized to receive maintenance prednisone (PRED) therapy or not (NOPRED) in addition to antilymphocyte globulin, cyclosporine, and azathioprine. Rejection episodes were initially treated with methylprednisolone pulses, and OKT3 monoclonal antibody was used to treat steroid resistant rejections that were verified by biopsy. NOPRED patients were declared protocol failures and returned to PRED therapy if they sustained 2 steroid-sensitive rejection episodes in the first 3 months or an OKT3-treated rejection at any time. Patient and graft survival for the LRD patients in both treatment categories were 100% at 12 months. Patient and graft survival for CAD recipients at one year was 94% and 83% (PRED) and 88% and 77% (NOPRED), respectively. Rejection episodes were more frequent (26 of 32 NOPRED patients vs. 19 of 35 PRED patients P = 0.02) and occurred earlier (4.5 weeks in NOPRED vs. 7.7 weeks in PRED patients) in patients not taking maintenance steroids. Rejection severity was also greater in the NOPRED group, as 15 OKT3-treated rejections occurred in that group whereas only 7 OKT3-treated rejections were observed in the PRED group (P = less than 0.01). The incidence of serious infection was similar in each group. Finally, protocol failure occurred in 40% of the LRD patients and 59% of the CAD patients. These data indicate that initiating maintenance therapy without PRED is safe but is attended by a greater risk of developing rejection. Because of this increased incidence and severity of early rejection episodes in NOPRED patients, we do not advise use of this immunosuppressive strategy in renal transplantation.

Effects of steroid withdrawal on posttransplant diabetes mellitus in cyclosporine-treated renal transplant recipients.

Posttransplant diabetes mellitus (PTDM) traditionally has been attributed to therapy with steroids--however, several lines of evidence suggest that cyclosporine also is diabetogenic. A retrospective review revealed that PTDM developed in 9 of 70 previously nondiabetic kidney transplant recipients (12.9%) maintained on prednisone, azathioprine, and CsA compared with 8 of 83 nondiabetics (9.6%) maintained on azathioprine and prednisone alone in an earlier era (P = NS). Among patients maintained on triple-drug therapy, complete withdrawal of prednisone was attempted in 7 renal transplant recipients with PTDM and in 1 recipient of a combined kidney-pancreas transplant who exhibited evidence of type II diabetes mellitus. Seven of the 8 patients were able to discontinue insulin or oral hypoglycemic agents within 4 months of discontinuing steroids. Mean glycohemoglobin level declined from 10.6 +/- 3.6% prior to steroid withdrawal to 6.0 +/- 1.3% within 1 month of steroid cessation, while mean CsA dose and trough CsA blood levels remained unchanged. In 2 patients, mild rejection episodes prompted a return to steroid therapy. Although CsA may be diabetogenic, evidence from this study suggests that withdrawal of steroid therapy is a safe and effective approach to the management of PTDM in patients subsequently maintained on CsA and azathioprine.

Laparoscopic right adrenalectomy after liver transplantation.

To our knowledge, laparoscopic right adrenalectomy has not been previously reported after orthotopic liver transplantation. The aim of this report is to demonstrate the feasibility of the laparoscopic approach in this technically challenging situation, and to outline some considerations unique to this clinical setting.

Withdrawal of steroids after renal transplantation--clinical predictors of outcome.

Withdrawal of steroid therapy in renal transplant recipients is associated with a risk of acute allograft rejection. To define clinical risk factors for rejection associated with steroid withdrawal, we analyzed the clinical characteristics of 107 patients with drawn from steroid therapy at various times after transplantation. Both univariate and multivariate analyses suggested that the timing of steroid withdrawal is an important predictor of steroid withdrawal failure. Withdrawal of steroids was successful in only 13 of 32 patients (41%) in whom prednisone was discontinued shortly after transplantation. In contrast, steroid withdrawal has been successful in 59 of 75 patients (79%) in whom prednisone was discontinued at least 6 months after transplantation. Black race and donor-recipient racial mismatch also were significant predictors of rejection associated with steroid withdrawal. In patients undergoing steroid withdrawal at least 6 months posttransplant, serum creatinine concentration also correlated independently with the risk of rejection. Neither age, sex, HLA match, pretransplant PRA, source of the allograft (cadaver vs. living relative), acute tubular necrosis, nor the presence of diabetes was predictive of the outcome of steroid withdrawal.

Variable effects of steroid withdrawal on blood pressure reduction in cyclosporine-treated renal transplant recipients.

The effects of complete withdrawal of steroid therapy on blood pressure and other clinical variables were studied in 58 renal transplant recipients subsequently maintained on azathioprine and cyclosporine; 76% of the patients were hypertensive prior to withdrawal of steroids. Cessation of steroids was accompanied by a significant decrease in mean arterial blood pressure and by a reduction in the number of required antihypertensive medications; however, changes in blood pressure were variable among individual patients. Previously normotensive patients exhibited little further decline in blood pressure. Multivariate analysis of the entire cohort of patients showed that the reduction in blood pressure accompanying steroid withdrawal was directly related to the prior severity of hypertension and inversely related to the dose of cyclosporine. We conclude that steroids play an important role in the pathogenesis of posttransplant hypertension in the majority of renal transplant recipients. Withdrawal of steroids generally is accompanied by reduction in blood pressure, but the benefit is greatest in previously hypertensive patients receiving relatively low doses of cyclosporine.

The prevalence of coronary artery disease in liver transplant candidates over age 50.

The prevalence of angiographically proven coronary artery disease (CAD) in adults with end-stage liver disease who undergo evaluation for liver transplantation is unknown; also it is unclear if cholestatic liver disease represents an independent risk factor. Patients with end-stage liver disease over age 50 having liver transplantation were studied using coronary angiography. Arterial stenosis was graded as normal, mild (< 30%), moderate (30 to 70%), or severe (> 70%). Risk factors for CAD were also assessed (male sex, smoking, hypertension, diabetes, family history of premature heart disease). Complications related to the angiography and decision making based on the findings were recorded. Thirty seven patients (23 females) with a median age of 61 years (range 50 to 71) underwent angiography. Thirteen patients (35.1%) had cholestatic liver disease. Thirty patients had no history of heart disease. The overall prevalence of severe coronary artery disease was 16.2% (95% confidence interval [CI] = 6.2% to 32.0%). No association was detected between CAD and cholestatic liver disease (P = 0.72). After eliminating seven patients with a prior history of angina (n = 1), myocardial infarction (n = 1), or coronary revascularization (n = 5), the frequency of moderate or severe CAD was 13.3% (95% CI = 3.8% to 30.7%). No association was detected between unsuspected CAD and cholestatic liver disease (P = 0.61). Diabetes was the most important risk factor for moderate or severe disease (P = 0.01). Patients without risk factors had significantly less CAD than the group as a whole regardless of the liver disease type (P = 0.02). Two patients experienced transient renal insufficiency after the angiography. Three patients with severe CAD were denied transplantation. We conclude that CAD represents a significant problem in patients over age 50 undergoing liver transplant evaluation. Cholestatic liver disease was not associated with a significantly higher prevalence of moderate or severe CAD in our population. Diabetes was the most predictive risk factor, and those without risk factors do not require extensive preoperative cardiac evaluation.

Kidney transplantation in patients aged sixty years and older.

Outcomes of renal transplantation were reviewed for 26 transplants performed in 25 patients 60 years of age or older between 1985 and 1989. Three grafts were from family donors and 23 were from cadaver donors. Twenty-one were first transplants and five were retransplants. Cyclosporine was used as primary immunosuppression and azathioprine and prednisone were administered to most patients. Overall patient and graft actuarial survival rates were 79% and 71%, respectively, at both 1 and 3 years. Patients (n = 14) free of both diabetes and cardiac disease (low risk) had 1- and 3-year patient and graft survival rates of 91% and 84%, respectively. Conversely, high-risk patients (n = 12) had patient and graft survival rates at 1 and 3 years of 67% and 58%, respectively. Early deaths (less than or equal to 6 months) were caused by sepsis (two patients) or cardiac events (three patients), and four of the five were in high-risk patients. Irreversible rejections and serious infectious complications were not as common as steroid-induced diabetes, which occurred in five patients. This experience suggests that kidney transplantation can be done safely and successfully in patients older than 60 years of age and should be the treatment of choice for low-risk patients in this category.

Pancreas transplantation in Ohio: a 15-year outcomes analysis.

BACKGROUND: Beginning in 1984, all pancreas transplantations performed in the state of Ohio have been tracked by the Ohio Solid Organ Transplantation Consortium (OSOTC). In this study the outcomes of these transplantations were compared across 3 eras to determine whether increasing experience has been beneficial. METHODS: Between July 1984 and December 1999, 765 kidney-pancreas (KPTx) and 76 pancreas only (Ptx) transplantations were performed. Outcomes measures for these 841 pancreas transplantations were compared over 3 eras, 1984 to 1989, 1990 to 1994, and 1995 to 1999. RESULTS: One-year patient survivals for KPTx patients were 87%, 92%, and 94% in the 3 eras, respectively. Graft survival at 1 year was also markedly improved between era 1 and era 3, increasing for PTx patients from 21% to 85% and for KPTx patients from 68% to 85%. Average waiting time increased from 132 to 318 days between era 1 and era 3. Conversely, average length of stay in hospital was significantly decreased from 34 to 18 days. The cost of the procedure, as measured by hospital charges, also decreased when compared in 1985 dollars as a technique to control for inflation. CONCLUSIONS: These data suggest that pancreas transplantation in Ohio has become a very successful and cost-effective therapeutic intervention for patients with type I diabetes with or without concomitant end-stage renal failure.

Liver resection using total vascular exclusion.

BACKGROUND: Total vascular exclusion (TVE) is a technique of liver resection that includes controlling both the suprahepatic and infrahepatic vena cava in addition to portal inflow at the time of parenchymal transection. We report a series of 61 liver resections in 60 patients using this technique. METHODS: A retrospective review of 61 procedures in 60 patients using TVE between 1990 and 1997 was carried out. No patient had cirrhosis. Parameters analyzed included age, gender, diagnosis, procedure, operative time, clamp time, intraoperative transfusion requirements, postoperative laboratory studies, length of stay (intensive care unit, ward), mortality, and morbidity. RESULTS: TVE was sustained hemodynamically in all patients. The mean age of the 34 men and 27 women was 56 years (+/- 15 years); 21% were older than 70 years. Eleven percent of the patients had benign lesions; 70% of the malignant tumors were metastatic. Seventy-five percent of the procedures were major or extended lobectomies. The mean operative and clamp times were 330 +/- 83 and 39 +/- 13.2 minutes, respectively; 68% had clamp times of < 45 minutes. The mean intraoperative red blood cell units was 1.45 +/- 1.93, with a range of 0 to 8 units; 48% required no transfusion and 80% received 2 units or less. There was 1 perioperative death for a mortality rate of 1.6%. The morbidity rate was 36%, which included 4 patients with postoperative liver dysfunction. Complications were not associated with transfusion but with clamp times exceeding 45 minutes. Liver dysfunction occurred with clamp times more than 60 minutes, particularly if the remaining liver parenchyma was histologically abnormal or the remnant was small. CONCLUSIONS: TVE is hemodynamically safe, even in patients older than 70 years. Blood loss during parenchymal transection is minimal; mortality and morbidity are low. The optimal clamp time is less than 45 minutes. Liver dysfunction is associated with clamp times exceeding 1 hour, particularly if the remaining parenchyma is abnormal or small.

Combined kidney and pancreas transplantation. A safe and effective treatment for diabetic nephropathy.

Seventeen patients with type I diabetes and diabetic nephropathy underwent combined kidney and pancreas transplantation. Mean age was 32 +/- 6 years and average duration of diabetes was 21 +/- 6 years. Transplantation was performed through bilateral iliac fossa incisions, and graft duodenocystostomy was used to achieve exocrine pancreas drainage. The actuarial patient and kidney survival rate was 92% and the pancreas survival rate, 88% (follow-up of 3 to 28 months). One patient with functioning grafts died at 10 months owing to a cardiac arrhythmia and one pancreas was "lost" to early thrombosis. Major morbidity was due to pancreas wound infection (53%) that required reoperation, but all patients have had subsequent healing of their wounds. Rehabilitation with return to meaningful activity has been accomplished by 15 patients. This experience demonstrates that combined kidney and pancreas transplantation can be performed safely and successfully.

Trends in bile duct injuries from laparoscopic cholecystectomy.

Bile duct injuries are a serious complication of cholecystectomy. Laparoscopic cholecystectomies (LC) were originally associated with an increased incidence of injuries. Patients referred to a tertiary center were reviewed to assess the trends in the number, presentation, and management. Seventy-three patients were referred over a 6-year period with a maximum of 17 patients referred in 1992, but the number has not declined substantially over time. The persistent number of referrals is a consequence of ongoing injuries. One third of injuries were diagnosed at LC, and the use of cholangiography has not increased. The number of cystic duct leaks has not decreased and they represent 25% of all cases. The level of injury has remained unchanged with Bismuth types I and II in 37% and types III and IV in 38%. Excluding patients with cystic duct leaks, 58% were referred after a failed ductal repair. Definitive treatment with biliary stenting was successful in 37%, and 34 patients (47%) required a biliary-enteric anastomosis. Complications occurred in 18 patients (25%) including seven with postoperative stricture or cholangitis. No biliary reoperations have been performed at a mean follow-up of 36 months.

Salvage of thrombosed forearm polytetrafluoroethylene vascular access grafts by reversal of flow direction and venous bypass grafting.

A technique is described for salvage of looped forearm polytetrafluoroethylene (PTFE) vascular access grafts that fail because of thrombosis due to cephalic vein outflow obstruction. It entails reversal of blood flow direction through the graft and construction of a new venous outflow in the medial upper arm. This procedure was performed in nine patients and, at the present time, has increased the graft life by an average of 6.2 months (range: 2 to 14 months) in eight. We conclude that this is a useful alternative to abandoning failed looped forearm PTFE grafts that have cephalic vein outflow obstruction.

Laparoscopic repair of recurrent ventral hernias.

Break down after repair of recurrent ventral hernias can exceed 50 per cent. Laparoscopic techniques offer an alternative. This study evaluated the efficacy of the laparoscopic approach for recurrent ventral hernias. A retrospective review on all patients with a recurrent ventral hernia who underwent laparoscopic repair at our institution from August 1995 to June 1997 was performed. Demographic, operative, postoperative, and follow-up data were collected. Thirty-one patients underwent an attempted laparoscopic ventral hernia repair. Sixteen were for recurrent hernias; 15 were successfully repaired laparoscopically. The patients were typically obese (mean body mass index, 30 kg/m2), had an average of 2.4 previous open repairs (range, 1-7), and six patients had previously placed intra-abdominal mesh. An average of 3.5 (range, 1-16) defects were found per patient with a mean total hernia size of 130 cm2 (6-480 cm2). In all cases, expanded polytetrafluoroethylene mesh (average, 299 cm2) was secured with transabdominal sutures. Postoperatively patients required an average of 19 mg of narcotics (MSO4 equivalent). Bowel function returned in 1.7 days. Length of stay averaged 2.0 days (1-4 days). There were two complications: cellulitis, which resolved with antibiotics, and skin break-down, which required mesh removal. With follow-up averaging 18 months (7-29 months), there is one recurrence; the case in which the mesh was removed. Laparoscopic repair of recurrent ventral hernia seems promising. Decreased hospital stays, postoperative pain, wound complications, and a low rate of recurrence are benefits of this technique.

Potent immunosuppression overcomes immunologic high-risk factors in recipients of cadaveric renal allografts.

With the introduction of more potent immunosuppressive regimens, increasing numbers of kidney transplant recipients traditionally viewed as being at high immunologic risk for rejection and graft loss have been accepted. These include recipients of multiple grafts, sensitized patients as measured by high panel reactive antibody (PRA), and patients with current warm B or historical positive crossmatches. Since November 1983, all recipients of cadaver kidneys have been treated with cyclosporine and prednisone. In addition, most also received a short posttransplant course of antilymphocyte globulin and long-term azathioprine. With these regimens, retransplantation, sensitization, current B-cell crossmatch and historical B- and/or T-cell crossmatch do not affect graft survival.

Treatment with OKT3 and cyclosporine for acute allograft rejection.

In summary, we believe that our experience with concomitant use of OKT3 and either reduced-dose CyA for treatment of renal allograft rejections or full-dose CyA therapy for treatment of liver allograft rejection is both safe and possibly more effective in reversing allograft rejection than use of the antibody alone. This strategy has also allowed us to use this MAb therapy without incurring the untoward consequence of the development of hightiter anti-OKT3 antibodies that could preclude its subsequent reuse.