In vitro monitoring of in vivo development of human anti-thymoglobulin antibodies by ELISA.

Thymoglobulin (rATG), polyclonal immunoglobulin, is prepared from rabbits immunized with human thymocytes. It is effective in prevention and treatment of renal allograft rejection. Human antibodies against antilymphocyte preparations can reduce efficacy by accelerating drug clearance or by inducing serum sickness. We developed an enzyme-linked immunosorbent assay (ELISA) to study posttreatment development of anti-rATG. In an Institutional Review Board-approved trial, we tested 101 allograft recipients for anti-rATG antibodies. Patients received rATG intravenously at 1.25 to 2.0 mg/kg/d for 2 to 14 days. Serum samples were obtained pretreatment and at weeks 1, 2, 4, 6, and months 3 and 6 post-rATG. ELISA plates were coated with rATG (10 microg/mL). Samples were diluted 1:100 and tested in quadruplicate. Positive samples were titrated. Horseradish peroxidase-conjugated (HRPO) affinity-purified goat anti-human immunoglobulin G (H&L) antibody reacted with bound human antibody. A chromagenic substrate for HRPO was added and optical density (OD, 490 nm) was read. An OD of twice the negative control was considered positive. Mean ODs of negative and positive controls were 0.113 +/- 0.030 and 1.042 +/- 0.196, respectively. Ten patients had detectable anti-rATG before rATG administration (1:100). Thirty-five of 101 patients (35%) developed anti-rATG antibody. Patients showed an initial positive anti-rATG antibody from days 8 to 59 after infusion and titers from 1:100 to 1:4000. In spite of rATG's postulated anti-B-cell activity, this study confirms that rATG induces sensitization at a frequency and titer seen with other xenogeneic antilymphocyte antibodies. Formation of such antixenoantibodies can have a negative impact on treatment response and hence warrant monitoring.

Histoplasmosis in renal allograft recipients. Two large urban outbreaks.

During two large outbreaks, ten episodes of histoplasmosis were documented in eight renal allograft recipients. Another episode occurred before the outbreaks. Associated infections with cytomegalovirus occurred in five patients and may have further impaired cellular immunity. Prolonged fever was the predominant clinical finding; and dissemination was observed in seven of our nine patients, including three with meningitis. Special stains of tissues and the histoplasmal complement fixation test provided useful diagnostic information rapidly, while cultures were eventually positive in seven patients. Treatment with amphotericin B resulted in prompt clinical improvement in all patients, but relapse occurred in two patients one year following therapy.

Histopathologic findings from 2-year protocol biopsies from a U.S. multicenter kidney transplant trial comparing tarolimus versus cyclosporine: a report of the FK506 Kidney Transplant Study Group.

BACKGROUND: This paper reports the histopathologic results of 2-year protocol biopsies from patients who were enrolled in the U.S. FK506 kidney transplant study . METHODS: Recipients of cadaveric kidney transplants were randomized to tacrolimus or cyclosporine therapy. Patients active in the trial at 2 years after transplantation were approached for a protocol biopsy. Biopsies were scored by the Banff classification in a blinded fashion by one pathologist. RESULTS: A total of 144 patients (41.3% of those active at 2 years) had a 2-year protocol biopsy performed; 79 patients were treated with tacrolimus and 65 patients were treated with cyclosporine. Evidence of acute rejection was found in seven (8.9%) of the 2-year biopsies in tacrolimus-treated patients and six (9.2%) cyclosporine-treated patients. Chronic allograft nephropathy was found in 49 (62.0%) tacrolimus biopsies and 47 (72.3%) cyclosporine biopsies (P=0.155). There were no apparent histopathologic differences between the tacrolimus and cyclosporine biopsies. The occurrence of chronic allograft nephropathy was significantly higher in patients who received a graft from an older donor (P<0.01), who experienced presumed cyclosporine or tacrolimus nephrotoxicity (P<0.001), who developed a cytomegalovirus infection (P=0.038), or who experienced acute rejection in the first year after transplantation (P=0.045). A multivariate analysis showed that nephrotoxicity and acute rejection were the most significant predictors for chronic allograft nephropathy. CONCLUSIONS: The occurrence of histologic acute rejection was rare at 2 years, confirming the absence of subclinical acute rejection in these late biopsies. A majority of the biopsies showed features consistent with chronic allograft nephropathy that was associated with acute rejection (particularly in cyclosporine-treated patients), nephrotoxicity, and cytomegalovirus infection in the first year. This suggests that nonimmunologic factors, such as drug-induced toxicity, may play an important role in chronic allograft nephropathy.

Therapy of acute cadaveric renal allograft rejection with adjunctive antithymocyte globulin.

A randomized and controlled study was conducted to evaluate the efficacy of adjunctive antithymocyte globulin (ATG) therapy for the treatment of the initial rejection episode in first cadaveric transplants. When compared to the control group (29), which received only standard antirejection treatment (SAT) of steroid pulsing and local irradiation, the adjunctive ATG treatment group (23) demonstrated significantly faster recovery rates (8.9 +/- 4.1 versus 6.9 +/- 3.7 days, P = 0.05, respectively) and better graft survival rates (62 +/- 9% versus 91 +/- 7%, respectively) after the first rejection. ATG treatment did not result in fewer subsequent rejection episodes than SAT but long-term allograft survival rates remained superior to controls for the entire 3-year study period. By avoiding ATG treatment in those patients who never experienced clinical rejection on maintenance immunosuppressive therapy, i.e., nonresponders (23 of 90), complications associated with excessive immunosuppression were minimized. The combined results of the non-responder group of patients and ATG-treated patients resulted in a 1-year patient survival of 97% and graft survival of 86%. These results suggest that the most efficacious use of ATG is therapeutic and not prophylactic in renal transplant patients.

In vitro effects of cyclosporin A on lymphocyte subpopulations. 1. Suppressor cell sparing by cyclosporin A.

The fungal metabolite, cyclosporin A, is a potent immunosuppressive compound. Experiments were performed in vitro with both human and nonhuman primate peripheral blood lymphocytes to study the effect of this agent on suppressor cell activity. Cyclosporin A did not affect the generation or function of concanavalin A-induced suppressor lymphocytes as measured by their ability to suppress thymidine uptake of lymphocytes in secondary cultures. No evidence of suppressor cell induction was noted by incubation of lymphocytes with only cyclosporin A. We conclude that, although cyclosporin A does not generate or induce suppressor cell lymphocytes, it does spare them, while inhibiting other subpopulations. This effect may create an imbalance in the immune system which results in profound suppression.

The immunosuppressive mechanism of azathioprine. I. In vitro effect on lymphocyte function in the baboon.

The effect of azathioprine on in vitro baboon lymphocyte function tests was evaluated using the mitogen stimulation test, the mixed lymphocyte culture test, the migration inhibition factor test, the cell-mediated lymphocytotoxicity test and the antibody dependent cell-mediated cytotoxicity test. It was found that azathioprine inhibited phytohemagglutinin and Concanavalin A stimulation at lower concentrations than those required to inhibit pokeweed mitogen stimulation. It inhibited the MLC reaction with as little as 0.2 mug/culture in the microculture system. Azathioprine had no effect on (a) the release of migration inhibition factor, (b) the cell-mediated lymphocytotoxicity assay if presensitized cells were used, and (c) the antibody-dependent cell-mediated cytotoxicity assay. However, azathioprine inhibited CML if it was added during in vitro sensitization and induction of killer cells. These in vitro results suggest that azathioprine inhibits those reactions which require cellular division.

Bacillary angiomatosis in a renal transplant recipient.

A case of bacillary angiomatosis infection presenting as a skin nodule in a renal transplant recipient was found. The patient was taking cyclosporine, prednisone, and mycophenolate mofetil at the time of presentation. The bacillary angiomatosis responded to 6 months of therapy with oral erythromycin.

A comparison of tacrolimus (FK506) and cyclosporine for immunosuppression after cadaveric renal transplantation. FK506 Kidney Transplant Study Group.

BACKGROUND: Tacrolimus (FK506), a macrolide molecule that potently inhibits the expression of interleukin 2 by T lymphocytes, represents a potential major advance in the management of rejection following solid-organ transplantation. This randomized, open-label study compared the efficacy and safety of tacrolimus-based versus cyclosporine-based immunosuppression in patients receiving cadaveric kidney transplants. METHODS: A total of 412 patients were randomized to tacrolimus (n=205) or cyclosporine (n=207) after cadaveric renal transplantation and were followed for 1 year for patient and graft survival and the incidence of acute rejection. RESULTS: One-year patient survival rates were 95.6% for tacrolimus and 96.6% for cyclosporine (P=0.576). Corresponding 1-year graft survival rates were 91.2% and 87.9% (P=0.289). There was a significant reduction in the incidence of biopsy-confirmed acute rejection in the tacrolimus group (30.7%) compared with the cyclosporine group (46.4%, P=0.001), which was confirmed by blinded review, and in the use of antilymphocyte therapy for rejection (10.7% and 25.1%, respectively; P<0.001). Impaired renal function, gastrointestinal disorders, and neurological complications were commonly reported in both treatment groups, but tremor and paresthesia were more frequent in the tacrolimus group. The incidence of posttransplant diabetes mellitus was 19.9% in the tacrolimus group and 4.0% in the cyclosporine group (P<0.001), and was reversible in some patients. CONCLUSIONS: Tacrolimus is more effective than cyclosporine in preventing acute rejection in cadaveric renal allograft recipients, and significantly reduces the use of antilymphocyte antibody preparations. Tacrolimus was associated with a higher incidence of neurologic events, which were rarely treatment limiting, and with posttransplant diabetes mellitus, which was reversible in some patients.

Persistence of cyclosporine after withdrawal of the drug in a patient with chronic liver transplant rejection. Role of the monoethylglycinexylidine test.

Patients with chronic rejection of liver allografts may show persistently high cyclosporine levels. This phenomenon may be due to a down-regulation of the P450 cytochrome system. The monoethylglycinexylidine test was useful in confirming this hypothesis.

Intraoperative placement of a Wallstent for portal vein stenosis and thrombosis after liver transplantation.

We report a case of orthotopic liver transplantation, in which portal vein thrombosis developed in the immediate postoperative period. Surgical thrombectomy and intraoperative placement of a large caliber Wallstent resulted in long-term patency. The unique feature of this case is the intraoperative placement of the stent via the inferior mesenteric vein under fluoroscopic guidance. The use of a large caliber (16 mm) stent obviated the need for postoperative anticoagulation.

Glomerular endothelial injury related to renal perfusion. A scanning electron microscopic study.

To elucidate abnormalities in renal morphology related to perfusion preservation, we examined kidneys perfused for 60 hr with a scanning electron microscope. In addition to tubular necrosis and fused glomerular visceral epithelial foot processes, we identified changes in the glomerular endothelial and arterial endothelial surfaces. The glomerular endothelium revealed fenestrae that were smaller and more irregular than normal, as well as abnormal bulbous projections. The arterial endothelium displayed striking degenerative changes. These abnormalities may account for the altered glomerular function and intravascular coagulation that occur in some kidneys preserved by lengthy perfusion.

Predictive value of enzyme-linked immunosorbent assay-detected IgG anti-HLA antibodies for pediatric renal allograft rejection.

BACKGROUND: Measurement of panel-reactive antibody (PRA) with an enzyme-linked immunosorbent assay using soluble HLA class I molecules (PRA-STAT) in adult renal transplant recipients predicted graft loss and rejection. We sought to confirm this finding in pediatric recipients, an immunologically distinct group. METHODS: The population consisted of 158 renal transplants in 146 patients (age range, 1-21 years). PRA was determined with PRA-STAT and microlymphocytotoxicity (CDC), using final cross-match sera. An elevated test was defined as > or =5% reactivity. Statistical analysis for rejection used the chi-square test and for graft survival used the log-rank test. RESULTS: Thirty-five patients (22%) had %PRA-STAT > or =5%, compared with 26 (16%) with %PRA-CDC > or =5%. The percentage with elevated %PRA-STAT was found to correlate with subsequent transplantations (first, 15%; second, 67%; third, 75%). Subsequent analyses utilized only the 136 primary recipients, of whom 20 (15%) had %PRA-STAT > or =5% and 16 (12%) had %PRA-CDC > or =5%. Elevated %PRA-STAT correlated with rejection at 3 months (65% vs. 36%), 12 months (84% vs. 50%), and 24 months (84% vs. 54%) (P<0.05). No association was found between elevated %PRA-CDC and rejection. Patients with %PRA-STAT > or =5% vs. %PRA-STAT <5% had graft survival at 1 year of 89% vs. 84%, at 2 years of 88% vs. 77%, and at 3 years of 61% vs. 72% (not significant). CONCLUSIONS: Use of %PRA-STAT > or =5% identifies pediatric recipients who are at increased risk for rejection and may benefit from more potent immunosuppression and/or closer monitoring of graft function.

Time course of glomerular endothelial injury related to pulsatile perfusion preservation.

To elucidate the time course of glomerular and arterial endothelial injury resulting from pulsatile perfusion preservation of human kidneys, we examined two kidneys, one at 16 and the other at 42 hr, for which no suitable recipient could be found. The scanning electron microscope revealed subtle changes at 16 hr in the filtration barrier. These included mild endothelial swelling with an increase in the appearance of bulbous processes, and elongated fenestrae. The visceral epithelial surface was normal as was the arterial endothelial surface. By 42 hr the glomerular endothelial surface displayed very prominent cytoplasmic ridges and clearly distorted fenestrae. The arterial endothelium exhibited a tendency to separate from the vessel wall. The proximal tubular epithelium revealed scattered loss of microvilli. These changes are similar in kind to, albeit less severe than, those described after 60 hr of perfusion. They may represent cell swelling following ischemia, or be the result of altered cell permeability engendered by low temperature. The possibility remains that such changes could be minimized by modifying the perfusate. Scanning electron microscopy provides a versatile tool in the study of vascular and other surfaces of tissues stored with perfusion preservation.

Prospective study of magnetic resonance imaging and SPECT bone scans in renal allograft recipients: evidence for a self-limited subclinical abnormality of the hip.

We recently reported that typical abnormalities of avascular necrosis (AVN) in magnetic resonance images (MRI) of the hips of asymptomatic renal transplant recipients whose plain radiographs are normal may improve spontaneously and even disappear completely. We present the results of serial bone scans, most of which were performed with single-photon emission computed tomography obtained over periods as long as 24 mo after transplantation in 72 of these patients. Three paired imaging studies (i.e., MR and bone scan performed within 30 days of each other) were available for each of these patients. In three patients, both the MR images and the bone scans showed changes consistent with bilateral AVN within 4 mo after transplantation. All three patients developed hip pain which was bilateral in two and unilateral in one. Two patients (three hips) required surgical intervention at which time AVN was found on pathologic examination of all three hips. None of the remaining 69 patients developed hip pain during the study. However, in nine patients whose MR studies were consistently normal, at least one bone scan was abnormal (13 hips). The presence of AVN was pathologically confirmed in each of the hips subjected to surgery. Where the imaging findings were identical to those in the asymptomatic patients as well as those in whom the imaging abnormality regressed, we suggest that the subclinical imaging abnormalities represent mild AVN, which is reversible in some cases. Since the process was identified in 10 hips by MRI and in 13 hips by bone scan, both studies are needed to detect subclinical AVN. This may be important if treatment of subclinical disease is clearly shown to prevent progression to symptomatic AVN.

Is there a role for Chlamydia pneumoniae in hemodialysis vascular access thrombosis?

Significant evidence suggests that Chlamydia pneumoniae has a major role in occlusive vascular disease. Vascular access thrombosis in chronic hemodialysis patients is a frequent problem; the underlying pathological state is stenosis caused by endothelial hyperplasia. There is presently no literature concerning C pneumoniae in vascular access thrombosis. We embarked on a study to evaluate the possible role of C pneumoniae in access failure. Ten consecutive patients with thrombosed polytetrafluoroethylene (PTFE) conduit arteriovenous fistulae undergoing surgical thrombectomy and revision were studied. We sought to detect C pneumoniae using both culture and polymerase chain reaction (PCR) methods. An excisional biopsy of the stenotic vein segment just above the anastomosis with the PTFE graft was obtained at surgery. Vein samples weighing at least 30 mg were aseptically placed in transport media and stored at 4 degrees C for up to 24 hours. The samples then were sonicated, inoculated in Hep-2 cell culture vials containing confluent monolayers, and passaged three times over 2 weeks. Detection was by direct fluorescent antibody staining. Both culture and PCR were performed in an active chlamydia research laboratory. None of the 10 samples was positive for C pneumoniae by culture or PCR. Based on our preliminary pilot study, we do not believe C pneumoniae has a major role in endothelial hyperplasia and consequent graft loss in the hemodialysis patients we studied.

Thrombofibrinous sheath occlusion of peritoneovenous shunts.

Thrombofibrinous sheath occlusion of peritoneovenous shunts is described in two case presentations. In reviewing the literature, we found only 17 other such cases mentioned. Most patients were seen initially with recurrence of ascites and only one with superior vena cava syndrome. A shuntogram documented a characteristic sheath in 17 patients, and relocation was the preferred treatment. Relocation was successful in eight of 11 attempts, but long-term follow-up data were not available. Histologic examination of the sheath in our first case study revealed recent and organized thrombus. This would help explain why only one of three patients was successfully treated with fibrinolytic agents.

Comparison of Minnesota antilymphoblast globulin and Upjohn antithymocyte globulin for induction immunosuppression of human renal allografts.

BACKGROUND: An analysis of heterologous polyclonal antisera in first renal transplants was continued after replacement of Minnesota antilymphoblast globulin (MALG) with antithymocyte globulin (ATGAM), testing the hypothesis that these are functionally equivalent drugs. METHODS: Sequential induction immunosuppression used MALG (20 mg/kg/day, n = 33) or ATGAM (15 mg/kg/day, n = 14), corticosteroids, azathioprine and cyclosporine. White blood cell, platelet, and T-cell subsets were measured. Percent of patients with and time to first rejection were determined. Anti-horse antibody was measured by enzyme-linked immunosorbent assay. Minimum follow-up after transplantation was 1 year. RESULTS: Human leukocyte antigen mismatch, peak and current panel reactive antibodies, age, gender, percent cadaver donors and diabetic recipients were similar. Depletion of CD2, CD3, CD4, and CD8 T-cell subsets and platelet and white blood cells was similar. Early renal function was better with MALG than with ATGAM (p = 0.005, ANOVA), but by 2 weeks the groups were similar. The percent of patients receiving MALG versus patients receiving ATGAM with cytomegalovirus (28 versus 50), anti-horse antibodies (50 versus 62), and rejection (58 versus 50) and the median day of first rejection (48 versus 47) were similar. Three grafts were lost. CONCLUSIONS: MALG and ATGAM are equally effective in eliminating T cells and preventing and delaying the onset of renal allograft rejection.

Tube jejunostomy in liver transplant recipients.

BACKGROUND: Nutritional support after liver transplantation most often uses intravenous hyperalimentation followed by nasoduodenal tubes until adequate intake is achieved. Because of difficulties with nasoduodenal tubes, we place jejunostomy tubes (j-tube) at the time of the transplantation, allowing immediate postoperative enteral nutrition. This review analyzes the complications of this procedure in transplant recipients. METHODS: J-tubes were placed in 108 of 119 adults who underwent liver transplantation between October 1989 and June 6, 1994. These patients were retrospectively reviewed for the type and frequency of j-tube-related complications. J-tube feeds with a semielemental formula were started within 24 to 48 hours after transplantation. RESULTS: Eighteen complications occurred in 16 patients. Six were mechanical obstructions of the j-tube because of kinking by the fascia. Six exploratory laparotomies were required, two each for infection, small bowel obstruction, or catheter displacement. Four other infections were treated by local incision and drainage or percutaneous drainage. One tube required surgical removal in the operating room. CONCLUSIONS: Tube jejunostomies can be safely placed at the time of liver transplantation with a low risk of serious complications. We recommend the routine use of j-tubes in patients receiving a liver transplant for the immediate posttransplantation institution of enteral nutrition.

Surgical considerations of continuous ambulatory peritoneal dialysis.

The surgical considerations pertaining to 173 continuous ambulatory peritoneal dialysis catheters were reviewed in 140 patients from 1979 through 1983. All catheters were inserted in the operating suite by an open technique. Local anesthesia was used in the majority of patients (59%). Catheter peritonitis was the most frequent complication, 228 episodes/2407 patient months. Twenty-three percent of the patients accounted for 51% of catheter-related peritonitis. Sixteen catheters were removed because of an inability to clear the infection. Intra-abdominal catastrophes were noted in four patients and differentiation from continuous ambulatory peritoneal dialysis peritonitis was based on serial examinations, bacteriologic cultures, and/or a progressive increase in free abdominal air. Surgically-related catheter complications were designated either early (less than 1 month) or late (greater than 1 month) in relation to catheter placement. Frequent early complications were mechanical flow problems and dialysate leaks. Five of 11 mechanical flow difficulties required catheter replacement while none of the dialysate leaks required surgical intervention, and all healed spontaneously. Common late complications included 35 tunnel infections, 23 of which were associated with peritonitis. Nine of these catheters (25%) were removed. Cuff extrusion was also associated with a high incidence (83%) of catheter attrition. Thirty-six patients underwent renal transplantation and in no instance did the catheter increase patient or renal allograft morbidity rates.

Acute lobar nephronia of renal transplant allograft.

Acute lobar nephronia (acute focal bacterial nephritis) has recently been recognized as an infectious process of the kidney. It is a radiologic diagnosis characterized as a nonliquifiable inflammatory renal mass associated with signs and symptoms of bacterial pyelonephritis. We present the successful management of a renal allograft recipient who had radiologic evidence of acute lobar nephronia within the graft six weeks after placement of an internalized ureteral stent.