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We compared our standard NIH (extended incubation) crossmatch (XM) with antihuman globulin (AHG) and flow cytometry crossmatches (FCXM) and correlated the results with primary cadaveric and retransplant graft survivals. In addition, we treated the XM sera with the reducing reagent dithioerythritol (DTE) to discriminate IgM from IgG immunoglobulin reactivity. For the 166 CsA-Pred-treated primary cadaveric renal allograft recipients the 1-year graft survival rate following an NIH-NEG XM was 81%. NIH-XM-NEG recipients who were also AHG-XM-NEG displayed an 82% 1-year graft survival as well. In contrast, NIH-NEG, but AHG-POS XM primary CAD recipients displayed a significantly reduced graft survival rate of 67%. Treatment of AHG-POS XM sera with DTE-delineated DTE/AHG-NEG and POS crossmatches associated with significantly different graft survivals of 83% and 0%, respectively, for these primary recipients. Flow cytometry XM results did not improve on the AHG-NEG or DTE/AHG-NEG XM primary graft survivals. These results were seen whether testing pre-Tx or historical (Hx) sera. For Re-Tx recipients an AHG-NEG XM resulted in significantly improved graft survival compared with the NIH-XM-NEG results. The overall 1-year graft survival rate for the 70 Re-Tx recipients studied was 64% (following a NEG pre-Tx NIH-XM). Re-Tx recipients with an AHG-NEG XM displayed an improved graft survival compared with NIH-XM-NEG recipients (77% vs. 64%, P less than 0.05) and with AHG-POS recipients (77% vs. 36%, P less than 0.01). However, treatment of Re-Tx, AHG-POS sera with DTE resulted in comparably poor graft survival rates of 31% and 50% for DTE/AHG-NEG and POS crossmatches, respectively. A FCXM did not improve on the results of Re-Tx graft survival following an AHG-NEG XM. These results were obtained whether testing pre-Tx or Hx sera. HLA matching, PRA, and the time the first Tx was lost did not influence the Re-Tx graft survival outcome following an AHG-NEG XM. Therefore, successful primary cadaveric renal allograft survival can be accomplished following either an AHG-NEG XM or an AHG-DTE-NEG XM. Re-Tx graft survival is significantly improved following an AHG-NEG XM. Re-Tx recipients with an AHG-POS XM who are either DTE/AHG-POS or -NEG display reduced graft survivals compared with AHG-NEG Re-Tx recipients.

We report that the mechanism of rapamycin (RAP) inhibition is synergistic, but nonidentical, to the mechanism of CsA inhibition. Like CsA, RAP inhibits T cell proliferation following mitogen (PHA) and/or alloantigen (MLR) stimulation. RAP levels of 100, 33, 11, 3.6, 1.2, and less than 1 ng/ml reduced PHA stimulation by 81%, 84%, 81%, 83%, 62%, and 33%, respectively, without cytotoxicity. The RAP concentration required to achieve 50% proliferative inhibition of either mitogen (PHA) or MLR assays revealed an interindividual variability of 5 pg/ml RAP (2 individuals), 1 ng/ml (3 individuals), and 100 ng/ml (2 individuals). Unlike CsA, RAP proliferative inhibition was not restricted to the G0 phase of the cell cycle. Addition of 100, 10, or 1 ng/ml RAP at the onset (G0), or 24 hr following cultivation (G1) similarly inhibited DNA synthesis by 42%, 42%, and 41% compared with 44%, 48%, and 47%, respectively. PWM-stimulated B cell proliferation was primarily RAP-sensitive during the G0 phase of the cell cycle. RAP at 100, 10, and 1 ng/ml inhibited B cell proliferation 46%, 51%, and 50% when added during G0 but only 15%, 20%, and 20% when added during G1. Generation of a cyclosporine-sensitive cytoplasmic activation signal, activator of DNA replication (ADR), was reduced by RAP. RAP reduction did not correlate directly with T cell proliferative inhibition (as does CsA). RAP-induced proliferative inhibition of 40% and 80% resulted in ADR inhibition of 16% and 33%. Proliferative inhibition was synergistically increased when CsA and RAP were used in combination, whereas ADR inhibition was only additively enhanced. Mechanistic disparity between RAP and CsA may potentiate clinical immunosuppression when RAP and CsA are used together.

The benefit of transplantation without prior dialysis might be contravened by the failure to develop possible immunologic disabilities associated with chronic uremia and dialysis. This study compares graft and patient outcome, cyclosporine toxicity, pharmacokinetics, rejection episodes, nutritional status, and social and vocational rehabilitation between a preemptive group of 85 patients transplanted without prior dialysis and a cohort of 84 demographically, temporally, and disease-matched recipients of renal transplants after a minimum of 6 months' chronic dialysis therapy. The groups were matched for donor type, gender, and age, as well as immunologic risk factors of HLA-mismatch and percent panel-reactive antibody. All patients received CsA and prednisone immunosuppression. There were only two differences between the cohorts. The preemptive group included more diabetic patients: 32 versus 15 (P less than 0.01). The control cohort included more recipients who had received any pretransplant transfusion: 55 versus 28 (P less than 0.001). Both of these factors (if having any impact) would be expected to reduce graft survival in the preemptive group. All patients in the study had a minimum follow-up of 1 year and over half of the recipients are beyond 40 months. The preemptive patients showed survival rates of 94, 93, and 91 percent at 1, 2, and 5 years. These rates were not significantly different from those of the control group, namely 96, 96, and 93 percent, respectively. The actuarial graft survival rates in the preemptive group of 83, 81, 76, 73, and 73 percent at 1, 2, 3, 4, and 5 years were not statistically different from the control group rates, namely 90, 81, 80, 77, and 76 percent. Preoperative blood transfusion or percent positive panel-reactive antibodies had no effect on postoperative outcome in either group. The incidence of CsA nephrotoxicity was 9.4 percent in the preemptive group, which was not statistically different from the 17.9 percent in the control group. The incidence of rejection episodes in the absence of patient noncompliance was comparable between the groups. Seven of the irreversible rejection episodes in the preemptive group were due to noncompliance, compared with none in the control group (P less than 0.001). Preemptive recipients were also more likely than control group patients to be employed fulltime both before transplantation (36 vs. 22, P less than 0.05) as well as after transplantation (38 vs. 20, P less than 0.01).(ABSTRACT TRUNCATED AT 400 WORDS)

While cyclosporine is recommended to be used only in conjunction with monitoring of its blood concentrations, the utility of these measurements in preventing treatment failure is not established. In a group of 52 patients trough levels and steady-state concentrations were monitored in serum and whole blood by specific (SP) and nonspecific (NS) assays (polyclonal radioimmunoassay, PR; fluorescence polarization immunoassay, FP; high-pressure liquid chromatography, HP). From as many as 10 determinations of trough level and steady state concentrations during the first 40 days after renal transplantation, the lowest measurement was selected. In the case of an acute rejection episode within that time period, only values until that event were considered. Trough level measurements in serum by PR/NS and by FP/NS and in whole blood by HP/SP were not significantly different between patients with and patients without rejection episodes. However, simultaneously measured steady-state values (serum/PR/NS and serum/FP/NS) were significantly lower in patients suffering from rejection (with rejection SS/serum/PR/NS mean = 127 ng/ml, SD = 41 ng/ml; without rejection mean = 163 ng/ml, SD = 60 ng/ml; P = 0.027, t test). This difference could not be demonstrated for steady state/whole blood/HP/SP measurements. A logistic regression analysis demonstrated that the probability of rejection can be decreased by up to 40% if steady state/serum/PR/NS or steady state/serum/FP/NS values never drop below 250 ng/ml early after renal transplantation.

Serum interleukin 6 (IL-6) levels were utilized as an immunologic marker of activation of T cells and macrophages in renal allograft recipients treated with a cyclosporine and prednisone immunosuppressive regimen. IL-6 concentrations were estimated in serum samples selected to correspond to similar timepoints in the clinical courses of renal transplant recipients suffering four types of events: group I, quiescent patients without rejection or infectious disease (n = 16, 147 samples); group II, patients with only rejection episodes (n = 26, 291 samples); group III, patients with only infectious episodes (n = 10, 87 samples); and group IV, patients with CsA-induced nephrotoxicity (n = 15, 117 samples). Serum IL-6 activity measured using an IL-6-dependent cell line (MH60.BSF-2) was specific for this lymphokine based upon the capacity of monoclonal anti-IL-6 antibodies to block target cell proliferation. The control group displayed uniformly elevated IL-6 levels during the first posttransplant day (mean 20.1 +/- 4.1 U/ml range 6.4-64 U/ml), thereafter decreasing by 10-14 days to a mean level of 3.4 +/- 0.9 U/ml (range 1.0-4.2 U/ml). The rejection group showed increased IL-6 levels ranging from 5.3 +/- 0.4 U/ml (range 1.0-64 U/ml) to 56.2 +/- 13.3 U/ml (range 10-300 U/ml, P less than 0.01), occurring at a mean of 2 days (range 0-10 days) before the diagnosis of rejection was established by clinical criteria. Interestingly, all three recipients treated with OKT3 and 5/11 treated with antilymphocyte globulin displayed further significant increases in serum IL-6 levels (OKT3: 46.0 +/- 12.9 U/ml; ALG: 34.6 +/- 7.8 U/ml) one day after inception of treatment. Five of 10 recipients displaying septic events showed elevated serum IL-6 activity--namely, 5.0 +/- 1.2 U/ml to 47.5 +/- 16.2 U/ml, beginning at a mean of 1.2 days before diagnosis. Contrariwise, recipients afflicted with CsA-induced nephrotoxicity displayed reduced IL-6 levels (mean = 1.4 +/- 0.18 U/ml). The ratio (IL-6 activity/CsA trough level) proved to be even more useful than the serum IL-6 level itself to discriminate acute rejection from nephrotoxicity--namely, 0.53 versus 0.006, respectively (P less than 0.01).

Rapamycin, an actinomycete macrolide lactone that inhibits cytokine-induced immunoactivation, and cyclosporine, an endecapeptide that prevents transcription of lymphokine messenger RNA, display mutually synergistic interactions in vitro and in vivo. Using the rigorous median-effect analysis to dissect the nature of immunosuppressive drug interactions, rapamycin significantly augmented the inhibitory effects of cyclosporine and/or dexamethasone upon human peripheral blood lymphocyte activation by phytohemagglutinin, anti-CD3 monoclonal antibody, and mixed lymphocyte reaction. Furthermore, the addition of rapamycin potentiated the activity of cyclosporine to reduce cytotoxic cell generation and precursor frequency during in vitro alloactivation, using cell-mediated lympholysis and limiting dilution analyses, respectively. Similarly, cyclosporine potentiated the inhibitory effects of rapamycin upon proliferation of IL-2 (CTLL-2) and IL-6 (MH60.BSF-2) lymphokine-dependent cell lines. Lineweaver-Burk plots of the Michaelis-Menton equation suggested rapamycin inhibits IL-2 signal transduction in competitive, and IL-6 signal transduction in noncompetitive fashion, suggesting distinctive components of the various cytokine-receptor mechanisms. In vivo the cyclosporine/rapamycin combination exerted synergistic immunosuppression of rejection reactions in rats toward heterotopic cardiac allografts, using concentrations at which drugs were individually ineffective. These observations suggest that cyclosporine and rapamycin may be combined at significantly reduced doses to achieve unprecedented levels of immunosuppressive efficacy.

The effectiveness of rapamycin (RAPA) was examined for heart, kidney, and small bowel allografts in rats. Untreated or vehicle only-infused Wistar Furth (RT1u) recipients rejected Buffalo (RT1b) heart allografts within a mean survival time (MST) of 6.5 +/- 0.5 and 6.3 +/- 0.5 days, respectively. In contrast, a 14-day continuous intravenous (i.v.) infusion by an osmotic pump of 0.08 mg/kg/day RAPA to WFu recipients prolonged BUF heart allograft survival to an MST of 34.4 +/- 12.1 days (P = 0.0001). There was a graded dose-response to 0.16 mg/kg (39.0 +/- 8.7 days; P = 0.0001), 0.32 mg/kg (55.7 +/- 3.3 days; P = 0.0001) and 0.8 mg/kg (48.0 +/- 3.6; P = 0.0001). Furthermore, intraarterial/intragraft but not i.v. infusion of 0.02 mg/kg/day prolonged BUF heart allografts--namely, an MST of 14.6 +/- 1.4 days versus 8.6 +/- 2.6 days (P = 0.0001), respectively. Local delivery doses of RAPA were about as effective as the same dose delivered i.v.: 0.08 mg/kg MST 37.0 +/- 18.3 days (P = 0.0001); 0.32 mg/kg, 40.0 +/- 3.9 days (P = 0.0001); and 0.8 mg/kg, 54.8 +/- 8.2 days (P = 0.0001). Systemic i.v. RAPA therapy with 0.08 or 0.8 mg/kg/day prolonged the survival of BUF kidney grafts in WFu recipients--namely, an MST of 52.7 +/- 42.7 (NS) and 90.2 +/- 62.4 (P = 0.001) days, respectively, versus an MST of 11.6 +/- 1.5 days in control WFu recipients only infused with vehicle. While normal WFu rats reject heterotopic BUF small bowel allografts within an MST of 10.0 days, a 14-day course of i.v. RAPA treatment significantly (P = 0.0001) prolonged small bowel allograft survival to an MST of 26.8 +/- 3.7 days.

The optimal measurement method and clinical application of the therapeutic drug monitoring of cyclosporine remain uncertain. At a workshop held at Hawk's Cay, FL, from January 14 to January 17, 1990, 57 scientists presented their latest research findings, either in formal papers or as discussants. Lively debate and discussion followed presentation of extant and new methodologies for drug measurements as well as multicenter validation studies: applications of trough-concentration monitoring in renal, hepatic, and bone-marrow transplants as well as in autoimmune disease; and alternative pharmacokinetic approaches to guide cyclosporine therapy. The process of inducing and maintaining optimal immunosuppression to facilitate graft success is a complex and often challenging task, requiring the combined expertise of multiple disciplines. Thus, the assembly of four of the groups essential to the transplant process--clinicians, laboratory scientists, the pharmaceutical company, and the manufacturers of cyclosporine measurement kits--provided a unique opportunity to evaluate therapeutic drug monitoring issues facing the transplant field. Here we present the major conclusions reached at the meeting, brief discussions of the study data on which they are based, and a summary of unresolved problems that will require further rigorous investigations. The Consensus Document was reviewed by all the workshop participants before we submitted this final manuscript.

Among the new techniques available for CyA monitoring, the FPIA offers the advantages of rapidity and simplicity. The present communication assesses the technical performance of this test in comparison with a 3H tracer-based PC-RIA for serum CyA levels using 971 samples obtained during the first 6 posttransplant months from 14 kidney transplant recipients. The FPIA evaluation included verification of CyA concentrations in manufacturer-supplied calibrators and controls by reference methods, determination of intraassay/interassay precision and accuracy, feasibility of specimen dilution and assessed assay sensitivity, and range of linearity. Comparison of FPIA with PC-RIA indicated that trough samples, when assessed by FPIA, averaged 1.3-fold greater than the PC-RIA, whereas non-trough FPIA measurements indicated similarity between the two methods. Although the two assays showed similar trends in most renal transplant recipients, two subjects demonstrated discrepancies, presumably reflecting the differing specificities of the polyclonal antibodies used in each assay. Thus, the FPIA appears to be a useful addition to CyA monitoring technology.

Though the high immunosuppressive efficacy of cyclosporine has revolutionized clinical transplantation, renal, hepatic, and neural toxicities limit its therapeutic potential. One approach to this problem is to combine CsA therapy with immunosuppressive agents that act synergistically with it, thereby permitting lower doses and mitigating drug-induced toxicity. The present study examines the in vitro interactions of various immunosuppressive agents--namely, 6-mercaptopurine (6MP), dexamethasone (Dexa), FK506, and Enisoprost (EP)--with CsA. These investigations dissect the impact of the drug combinations in inhibiting 3H-thymidine incorporation into DNA by normal human peripheral blood lymphocytes activated by mitogens or by alloantigens in mixed lymphocyte culture responses. The median-effect equation was used to analyze the dose-effect relationships of immunosuppressive drugs either alone or in combination. The interactions were quantified by calculation of combination indices (CI). CI values less than 1, greater than 1, and equal to 1 represent synergistic, antagonistic, and additive interactions, respectively. In combination with CsA, 6MP showed modest and Dexa profound synergism, while EP displayed modest and FK506 marked antagonism. These findings confirm the clinical impression of in vivo synergism between CsA and Dexa. They suggest that in vitro analysis of drug interactions may distinguish synergistic drug combinations potentially applicable to clinical practice.

The addition of 3M KCl-extracted donor antigen (HAg) to immunosuppressive therapy with 16 Gy total lymphoid irradiation produces a significantly higher fraction of Wistar-Furth (WFu) recipients displaying indefinite survival of heterotopic buffalo (BUF) heart allografts, namely 80 versus 20%. The experiments presented herein analyzed the direct activity as well as estimated the potential precursor numbers at 1 and 3 months in treated recipients. At 1 month post-TLI/HAg therapy, recipients showed reduced proliferative responses in mixed lymphocyte reactions (MLR) in a specific pattern toward donor but not third-party stimulators. Both TLI/Graft and TLI/HAg/Graft groups showed a higher frequency of BUF antigen-directed T-cytotoxic cells (fTc) than TLI-treated, but nontransplanted, WFu hosts. In addition, the TLI/HAg group alone displayed alloantigen-specific suppressor cells that suppressed the MLR proliferative responses of normal spleen T cells against donor, but not third-party, alloantigens. At 3 months postirradition, both TLI/Graft and TLI/HAg/Graft groups displayed variable MLR proliferative responses toward donor and third-party alloantigens. Whereas nontransplanted, TLI-treated WFu rats recovered their fTc to normal levels at 3 months, the TLI and TLI/HAg treated recipients bearing functional heart allografts demonstrated significantly decreased splenic fTc. These results show that reduced numbers of cytotoxic cell precursors may afford more reliable indices of prolonged heart allograft survival than MLR responses. The observations suggest that TLI/HAg transplant hosts display both reduced cytotoxic precursors and activated suppressor elements.

While CsA has improved renal-allograft survival rates in the first 2 years compared with Aza, Terasaki's multicenter study (1) failed to show any difference in long-term graft survival in CsA-Pred versus Aza-Pred-treated recipients. The present study examines the long-term graft-survival rates at a single center using CsA immunosuppression and seeks to discern the causes of 58 graft losses among 343 patients with functioning grafts beyond 2 years posttransplantation. The 6-year primary and cadaveric actuarial graft survival at this institution is 59% with a graft half-life of 10 years, which is better than the 40% and 7.7 years, respectively, reported by Terasaki (1) for primary cadaveric recipients on Aza-Pred. It is also better than the 41%, 6-year survival and 5.5-year half-life for primary cadaveric recipients treated with CsA-Pred as reported in the multicenter study. (1) Less experience with the use of CsA may explain the latter comparison. Primary LRD grafts at this institution (2/3 haploidentical) have a 6-year actuarial survival of 77% and a half-life very closely approximating that of HLA-identical LRD grafts under Aza (23.4 years). These results demonstrate that CsA mitigates the effects of HLA incompatibility to reduce graft survival. The most common cause of graft loss beyond 2 years was chronic rejection (36.2%) followed by noncompliance (27.6%). Patient deaths resulted in 13 of the 58 graft losses; most of the deaths were related to cardiovascular diseases. Only 3 patients died from causes that could be attributed to CsA immunosuppression; 2 from sepsis and 1 from viral hepatitis. Acute rejection caused 8.6% of the graft losses on continuous CsA therapy. When immunologic risk factors were analyzed, the entire graft-loss group had a significantly higher proportion of retransplant patients than the graft-survival group (P less than 0.005), suggesting that prior transplantation imposes a higher risk for graft loss not only acutely but long term as well. However, retransplanted patients were significantly less likely to lose their grafts because of noncompliance (P less than 0.005). Male patients were found to be significantly more noncompliant.

The cellular mechanisms of unresponsiveness induced with a combined KCl-extracted donor antigen (HAg) and CsA regimen were dissected by limiting-dilution (LD) assay. While untreated Wistar-Furth (WFu, RT1u) rats reject Buffalo (BUF, RT1b) heart allografts within a mean survival time of 6.6 +/- 0.5 days, recipients treated with 3 cycles of CsA alone (-1,0,1; 7,8,9; 15,16,17) maintained BUF heart allografts up to an MST of 22.5 +/- 8.9 days. When CsA was combined with BUF HAg (-1), BUF heart survival was further prolonged up to an MST of 34.2 +/- 6.6 days, while third-party BN HAg was ineffective (MST of 21.5 +/- 2.1 days). On day 10 postgrafting, the frequency of T cytotoxic cells (fTc) within the splenic pan-T-cell population was 1:1437 +/- 301 in CsA and 1:1087 +/- 438 in CsA/HAg treated recipients. In contrast, on day 30 postgrafting, both CsA and CsA/HAg treated WFu rats bearing functional BUF hearts showed within their splenic pan-T-cell populations a profound decrease in fTc to 1:2966 +/- 824 with CsA alone and to 1:4946 +/- 938 with CsA/HAg treatment. In contrast, both untreated WFu rats who rejected BUF heart allografts and CsA-treated WFu recipients who had rejected their BUF heart allografts on day 20 displayed an increased fTc to 696 +/- 243 and to 1:1169, respectively, when examined at day 30 postgrafting. Additionally, both the W3/25+ and OX8+ T cell subsets specifically suppressed the proliferative response of normal WFu T cells against BUF and, to a lesser degree, third-party BN irradiated stimulators. Thus, CsA-treated animals develop a potent specific-suppressor mechanism that is augmented by pretreatment with donor soluble antigen. This suppressor activity may decrease the frequency of alloantigen-specific Tc cells and thereby prolong the survival of BUF heart allografts.

We compared our standard NIH (extended incubation) crossmatch (XM) with antihuman globulin (AHG) and flow cytometry XMs and correlated the results with rejection episodes and graft survivals. For 89 CsA-Pred, primary renal allograft recipients, AHG and/or FCXM results did not improve on the NIH-XM-negative (NEG) graft survival results, whether testing pretransplant or historical (Hx) sera. Similarly, there was no association of a positive (POS) AHG or FCXM with increased rejection episodes in these primary recipients. However, for retransplant (Re-Tx) recipients a neg AHG or FCXM did discriminate fewer rejections and an improved graft survival compared with the NIH-XM-neg. results. The overall one-year graft survival for the 47 Re-Tx recipients studied herein was 66% (based on a neg pre-Tx NIH-XM). Pre-Tx AHG-NEG, Re-Tx recipients displayed an improved graft survival compared with NIH-XM NEG recipients (77% vs. 66%, P less than 0.05) and with AHG-POS recipients (77% vs. 47%, P less than 0.05). Similarly, pre-Tx, FCXM-NEG, Re-Tx recipients displayed improved graft survivals compared with NIH-XM-NEG recipients (83% vs. 66%, P less than 0.05) and FCXM-POS recipients (83% vs. 48%, P less than 0.05). Re-Tx recipients displaying a POS AHG and/or FCXM experienced a significantly greater number of rejections than NEG-XM recipients (P less than 0.05, respectively). The AHG and FCXM results correlated with rejections and graft survivals whether testing pre-Tx or Hx high-PRA sera. Re-Tx recipients who were AHG-XM-NEG but FCXM-POS, experienced more rejection episodes than recipients who displayed a negative XM reactivity for both AHG and FCXM (P less than 0.02), but with no resulting differences in graft survival. HLA matching, pre-Tx blood transfusions and PRA did not impact on these crossmatch and graft survival results. Use of AHG and/or FCXMs for Re-Tx, but not primary, recipients should help to improve graft survival for these high-risk recipients.

The measurement of areas under the concentration-time curve (AUC) was recently introduced as an alternative to trough level monitoring of cyclosporine therapy. The AUC is divided by the oral dosing interval to calculate an average concentration. All measurements are performed at clinical steady state. The initial evaluation of AUC monitoring showed advantages over trough level monitoring with concentrations of cyclosporine measured in serum by the polyclonal radioimmunoassay of Sandoz. This assay technique is no longer available and the following assays were performed in parallel during up to 173 AUC determinations in 51 consecutive renal transplant patients: polyclonal fluorescence polarization immunoassay of Abbott in serum, specific and nonspecific monoclonal radioimmunoassays using 3H and 125I tracers in serum and whole blood, and high performance liquid chromatography in whole blood. Both trough levels and average concentrations at steady state measured by those different techniques were significantly correlated with the oral dose. The best correlation (r2 = 0.54) was shown by average concentrations measured in whole blood by the specific monoclonal radioimmunoassay of Sandoz (3H tracer). This monitoring technique was also associated with the smallest absolute error between repeated observations in the same patient while the oral dose rate remained the same or was changed. Both allegedly specific monoclonal radioimmunoassays (with 3H and 125I tracer) measured significantly higher concentrations than the liquid chromatography. (ABSTRACT TRUNCATED AT 250 WORDS)

Within the cascade of intracellular activation signals triggering T lymphocyte effector response to alloantigen is a cytoplasmic protein, ADR, that activates DNA replication in isolated nuclei. Quantitative changes in ADR (exclusive to activated cells) regulate cell cycle progression and may indicate changes in intracellular proliferative control. The present communication documents that inhibition of ADR activity reflects the in vitro immunosuppressive effects of Cyclosporine. CsA inhibition of both proliferation and generation of ADR was concentration-dependent and occurred only in the G0 phase of the cell cycle. Drug addition did not affect G1 cells. ADR generation was inhibited both by CsA and by PGE2 alpha, possibly via effects on calcium-dependent activation pathways confined to G0/G1 transition. On the other hand, ADR generation was not inhibited by the immunosuppressive agents 6-mercaptopurine, Enisoprost (a PGE1 analog), or FK506. ADR activity was sensitive to aprotinin, which typically inhibits serine proteases. Using an enzymatic assay to quantitate serine protease activity (SPA) following PHA stimulation revealed that ADR content inversely correlated with SPA: ADR was only present in activated cells; SPA was highest in resting cells and decreased after PHA stimulation. The PHA-induced fall in SPA activity was inhibited by CsA, consistent with the failure to generate ADR. Like ADR, SPA was sensitive to PGE2 alpha and quantitatively unaffected by 6-mercaptopurine, Enisoprost, or FK506. Thus, ADR and SPA may represent opposing components of a cytoplasmic signaling cascade the balance of which reflects the level of immunosuppression, and thus represents a focus for in vitro evaluation of the immunologic response of allografted patients to cyclosporine.

Of 704 renal transplant recipients receiving long-term cyclosporine immunosuppression, 71 patients experienced proteinuria greater than 1 g/24 hr beyond the first month posttransplant. Eight patients displayed transient proteinuria, defined as lasting less than 3 months. In most cases this condition was attributed to biopsy-proved acute rejection. The transient proteinuria cohort experienced good graft outcome--namely, 87.5% one-year and 52.5% five-year actuarial graft survivals, which was similar to that observed in patients without proteinuria. In contrast, 52.4% of the 63 patients with nontransient proteinuria experienced graft loss within a median time of 6.1 months. The one- and five-year actuarial graft survivals in patients with nontransient proteinuria were 75.3% and 37.5%, respectively. Among the 63 patients with nontransient proteinuria, histopathologic diagnosis included chronic rejection in 19, transplant glomerulopathy in 14, acute rejection in 9, glomerulonephritis (GN) in 7 including 2 cases of membranous GN, and nonspecific interstitial fibrosis in 10 cases. Despite the overall poor prognosis for graft survival among the entire cohort of patients with nontransient proteinuria, the seven with allograft GN maintained prolonged graft function. They showed an 83.3% five-year actuarial graft survival versus 31.2% in patients with other causes of proteinuria (P = 0.043). These results suggest that posttransplant proteinuria in CsA-treated renal transplant recipients arises primarily as a consequence of allograft rejection and portends a poor graft outcome.

It has been reported that initial cyclosporine levels over 400 ng/ml posttransplantation result in an increased incidence of delayed graft function (DGF). Several studies have shown early graft function to be a major determinant for long-term graft survival. Continuous intravenous infusion (CIVI) has been employed to induce immunosuppression establishing therapeutic drug levels while minimizing toxicity in renal allograft recipients. This study examines the impact of the achieved serum CsA steady-state concentration (Css) levels upon transplant outcome in 228 patients given CsA by CIVI. In spite of administration of a specific drug dose, interindividual variation in elimination rates yields a broad range of Css levels. Six groups were stratified by CsA Css levels: group A 0-75 ng/ml, group B 76-100 ng/ml, group C 101-150 ng/ml, group D 151-200 ng/ml, group E 201-250 ng/ml, and group F greater than 250 ng/ml. Group A showed a significantly lower age and greater incidence of rejection at 0-10 days. Group F had significantly higher incidences of nephrotoxicity, hepatotoxicity, and delayed graft function. The findings suggest that the antirejection Css threshold for CsA may be at least 75 ng/ml, and the toxicity threshold above 250 ng/ml. Controversy exists about whether CsA influences the incidence of DGF, therefore risk factors for DGF were examined among the groups stratified by CsA Css levels. While cold ischemia time for all 228 patients as a group was highly correlated with DGF (P less than 0.001), neither cold ischemia time nor donor age was significantly different among the groups. There does appear to be a synergistic effect between CsA Css and CIT, since the incidence of DGF was significantly higher when the cold ischemia time was 21-24 hr and CsA Css greater than 200 ng/ml. Long-term graft function did not appear to be affected by early CsA Css levels. The Css of 100-250 ng/ml appears to achieve a satisfactory outcome with a 19.5% incidence of rejection within 10 days, 29.7% DGF, and 5.1% nephrotoxicity. Only 118/228 patients (52%) in this study achieved that range despite a fixed low CIVI of CsA. Thus potential renal allograft recipients may benefit from a pretransplant pharmacokinetic study to predict the proper CIVI dose.

The causes of variability in cyclosporine (CS) clearance (CL) are mostly unknown. The pharmacokinetics of CS were studied in 30 adult uremic patients after single intravenous and oral doses by analyzing serial concentrations in serum by radioimmunoassay (SR) and in whole blood by radioimmunoassay (WR) and high pressure liquid chromatography (WH). Bioavailability (F) and CL were calculated by noncompartmental models and were significantly different depending upon the assay method except for FSR = FWR: FSR = 43.2 +/- 21.7%; FWR = 43.5 +/- 18.5%; FWH = 36.4 +/- 17.3%; CLSR = 849 +/- 363 ml/min; CLWR = 380 +/- 156 ml/min; CLWH = 559 +/- 174 ml/min. The age of the patients and parameters describing body size such as weight, surface area and percent of ideal weight were not correlated with CL. The height of the patients correlated with CLWH but not CLSR or CLWR. Parameters responsible for CS binding in blood such as cholesterol, triglyceride, hemoglobin concentration or hematocrit did not explain variability in CL. Of the factors indicative of liver function alanine transaminase activity but not aspartate transaminase, lactate dehydrogenase, alkaline phosphatase activity nor total bilirubin concentration in serum was correlated with CL. F was not correlated with any of the demographic factors except for alanine transaminase. None of the significant correlations explained enough of the variability to afford a reliable prediction of CL or F.

Recent studies have demonstrated that hepatic dysfunction, induced by experimental biliary ligation (EBL), impairs lymphocytic responsiveness to PHA stimulation in vitro and to cellular antigens in vivo. This suppression appears to be selective for T-cell mechanisms while B-cell-mediated functions remain intact. The purpose of this study was to determine whether coexisting hepatic insufficiency could exert a protective effect on vascularized or nonvascularized allograft survival in the transplanted recipient. Female Wistar-Furth (Rtlw) 225 g rats were assigned randomly to three groups: EBL, sham operation (Sham) and normal control (NC). Fourteen days following operation animals received heterotopic cardiac or skin allografts from Buffalo (Rtlb) donors. Cardiac and skin graft survival was determined daily, rejection was confirmed histologically, and technical failures were omitted from analysis. Allograft survival was expressed as median survival time +/- SEM. Serum total bilirubin (mean +/- SEM) was significantly elevated at Day 14 in EBL animals compared to Sham and NC groups (15.1 +/- 1.0 vs 0.1 +/- 0 and 0.2 +/- 0.1 mg/dl, respectively, P less than 0.01). Median cardiac allograft survival time by Probit was 10.6 +/- 2.6 vs 5.6 +/- 0.7 and 6.0 +/- 0.9 days, respectively (P less than 0.03). Skin graft survival (mean and range) was similar in all groups. These results demonstrate that EBL in the rat suppresses T-cell function and significantly prolongs vascularized allograft survival, but not skin allograft survival across the Rtl histocompatibility barrier. The mechanism whereby coexisting hepatic dysfunction exerts a protective effect on vascularized allograft survival warrants further investigation.

The suggestion that hypertension is more prevalent in renal allograft recipients receiving cyclosporine (CyA), particularly those displaying nephrotoxicity, was tested by reviewing 200 patients' courses, including 92 cadaver (CAD) and 58 living-related (LRD) transplants using CyA and prednisone immunosuppression, and 19 CAD and 31 LRD transplants using azathioprine (Aza) and prednisone, all of whom had at least 1 year posttransplant complete outpatient follow-up. Both groups had a mean age of 33 years with a similar distribution of renal failure etiologies. Renal function was significantly impaired in the CyA group at all intervals (P less than .001, t test). The prevalence of hypertension was higher in the CyA group at all intervals, becoming significant at 12 (P less than .01) and 24 (P less than .05) months following transplantation (chi 2). While there was only a significant difference in mean diastolic BP at 12 months (P less than .05, t test), the mean number of antihypertensive and/or diuretic medications was significantly greater in the CyA group at 1 and 6 months (P less than .001) and at 12 months (P less than .01). By 24 months, the mean number of all antihypertensive and/or diuretic medications was no longer significantly different. However, the antihypertensive and diuretic requirements of the CyA group diminished with time, suggesting that the hypertension is not progressive if the CyA serum trough levels are maintained in the nontoxic range (less than 200 ng/mL).

The anterior extraperitoneal approach was compared to the flank approach for living donor nephrectomy in a series of 36 familial donors. The former procedure (23 cases) not only afforded superior visualization of renal vessels but also was probably at least as safe as the latter (13 cases) for donors with risk factors of obesity, age more than 45 years and pulmonary disease. Anterior extraperitoneal nephrectomy appears to be indicated for donors with multiple renal arteries and skeletal deformities, including thoracolumbar arthritis.

In order to assess the impact of demographic factors on serum levels of cyclosporine (CsA) estimated by radioimmunoassay (RIA) in renal allograft recipients, 493 pharmacokinetic studies were performed in 212 patients. Neither the presence of diabetes mellitus nor the CsA dosing frequency affected the measured pharmacokinetic parameters. Age over 45 years led to slower CsA clearance with resultant increase in maximum serum concentration (Cmax) per administered milligram, and increased volume of distribution. Female patients showed more rapid drug clearance, but greater volume of distribution. Concomitant hepatic impairment reduced drug clearance, increasing the area under the curve (AUC) per administered milligram of drug, and the Cmax. Patients treated with a rapid steroid taper showed a shorter half-life and lower Cmax than those receiving a slow steroid taper. Nephrotoxicity was associated with increased AUC per administered mg, while patients with acute tubular necrosis requiring dialysis showed poorer drug absorption, lower Cmax, and longer time to peak. The only effect of cimetidine administration was a slightly shortened time to peak. Serial analyses posttransplant in 17 patients suggested a tendency toward improved drug absorption with no effect on other parameters. These studies demonstrating the significant impact of demographic factors thus afford a basis on which to predict the trend of anticipated CsA levels as measured by RIA in renal allograft recipients.

Cardiac transplantation has evolved from an experiment to an accepted therapy for severe heart failure. Increasing competition for donor organs mandates a greater emphasis on selection and timing for transplantation and paradoxically forces more reliance on aggressive medical therapy for all patients after evaluation. The growth of recipient and donor pools may enhance the opportunity for assessing histocompatibility, for which distinguishing between autoantibodies and human leukocyte antigen-determined reactivity is important, and some general nonresponders may be detected. Therapy with cyclosporine has improved the outcome after transplantation, but further refinement is needed, perhaps with pharmacologic synergy, to minimize nephrotoxicity and maximize specific immunosuppression. Survival is more than 80% at 1 year, after which the incidence of acute rejection and infection declines and accelerated atherosclerosis becomes prominent. Although resuming employment is not always possible, the overall quality of life is excellent after cardiac transplantation.

The effect of splenectomy on neoplastic outgrowth was examined prior to and after implantation of methylcholanthrene-induced C3H/He murine tumors. Splenectomy performed 12 days before tumor inoculation did not affect the tumor outgrowth, however, both splenectomy and sham operation performed shortly before tumor inoculation resulted in significant tumor facilitation compared with the non-operated group, suggesting that this accelerated tumor was not related to the presence or absence of splenic tissue, but rather to systemically-induced immunosuppression. While splenectomy performed 6, 9, 12 days after tumor inoculation did not alter the tumor growth, splenectomy performed early (3 days) or late stage (20 days) after tumor cell challenge revealed a retarded neoplastic outgrowth, compared with the sham operated group. These results suggest that splenectomy in very early and late stages of tumor-bearing host may be effective for tumor treatment.