Alpha interferon combined with ribavirin potentiates proliferative suppression but not cytokine production in mitogenically stimulated human lymphocytes.

The improved clinical outcome observed among patients with hepatitis C treated with the combination of alpha interferon (IFN) and ribavirin (RBV) is presumed to result from immunomodulation and viral inhibition. However, the impact of the drug combination upon lymphocyte activity is unknown. The present study evaluated the effects of IFN and RBV, singly and in combination, upon proliferation, cell cycle sensitivity and cytokine elaboration following PHA stimulation of lymphocytes. Two formulations of IFN, interferon-a-2b (IFN-2b) and interferon-a-con-1 (CIFN), were included. Titration of each drug over a wide range of concentrations showed dose dependent proliferative suppression without cytotoxicity. Proliferation was suppressed 57-99% (P<0.001) by IFN-2b (10(5)-10(7) IU/ml), 41-74% (P<0.001) by CIFN (1.5-150 ng/ml), and 10-94% (P<0.001) by RBV (0.5-50 microg/ml). Isobologram analysis showed that the interaction between IFN-2b and RBV on proliferative suppression was additive. In contrast, the interaction between CIFN and RBV was weakly antagonistic. Proliferative suppression by both the IFNs was cell cycle restricted. IFN-2b and CIFN added at the onset of PHA stimulation (G0/G1) versus 24 h later (S phase) inhibited proliferation by 50 versus 5%, respectively (P<0.05). The onset of IFN resistance correlated with a 50% reduction (P<0.05) in IFN receptors on the cell surface. In contrast, RBV caused equivalent proliferative suppression (P=NS) when added at any time during PHA activation. Cytokine secretion after 24 h of PHA stimulation showed that IFN-2b versus CIFN increased the secretion of IL2, TNF and gamma IFN by 4.5-, 4.1- and 8.3-fold (P<0.005) versus 1-, 1.9- and 1.9-fold (P<0.05), respectively, above control levels. Neither IFN affected IL10 secretion. RBV, singly and in combination with IFN, had no impact on cytokine expression (P=NS). This study identifies several potential mechanisms by which the combination of IFN and RBV may exert a more potent effect upon cellular immunity than either agent alone and shows that different formulations of IFN may have non-identical effects upon lymphocyte responses.

A prospective randomized trial of mycophenolate mofetil with neoral or tacrolimus after orthotopic liver transplantation.

BACKGROUND: The success of liver transplantation in this decade has become the stimulus to extend the donor and recipient pool. Reducing early posttransplant morbidity to maintain our success, as we expand our frontiers, has led us to focus on balanced testing of multidrug immunosuppression regimens. METHODS: A prospective trial in orthotopic liver transplantation using Mycophenolate Mofetil and an identical steroid taper with randomization of patients to Neoral (N) or Tacrolimus (FK) is the basis of this report. This was an intent-to-treat study designed to compare the 6-month primary endpoints of rejection and infection and to compare the 6-month secondary endpoints of liver function, renal function, bone marrow function, hypertension, and serum cholesterol levels. RESULTS: Ninety-seven patients completed the 6-month follow-up period (N=49, FK=48). The actual 6-month patient and graft survival rates were 98% and 94%, respectively. There was no difference in the number of patients with rejection episodes (N=11, FK=8) (P=0.61). There were 24 infections (3 cytomegalovirus) in the FK group and 30 infections (9 cytomegalovirus) in the N group. The cholesterol levels at 6 months were not significantly different (P=0.07) between the groups. The other secondary 6-month endpoints were not significantly different, except total bilirubin, which was lower in the FK arm (P=0.02). CONCLUSIONS: The use of Mycophenolate Mofetil with N or FK and an identical steroid taper after orthotopic liver transplantation is associated with excellent graft and patient survival, and at 6 months, only 191% of the patients experienced rejection, with a 48% overall infection rate.

The impact of a positive crossmatch upon outcome after liver transplantation.

Recent reports have shown that liver allografts transplanted against a positive lymphocytotoxic crossmatch (CDC+) are susceptible to an increased frequency of rejection, and decreases in patient and graft survival. The implication of a positive flow cytometric crossmatch (FCXM+) in liver transplantation remains controversial. The purpose of this study was to determine what impact a pretransplant IgG crossmatch due to CDC+ or FCXM+ had upon the clinical outcome following liver transplantation. Preoperative crossmatch status was determined prospectively in 110 consecutive liver transplants performed between July 1991 and January 1995. Allografts were divided into three groups: negative crossmatch (NXM), positive flow cytometric crossmatch FCXM+, and positive lymphocytotoxic crossmatch CDC+. Crossmatch status did not impact patient or graft survival. Actuarial patient survival was similar between groups at 12 months (88% vs. 95% vs. 92%, NXM vs. FCXM+ vs. CDC+) and 24 months (81% vs. 93% vs. 92%, NXM vs. FCXM+ vs. CDC+) (P=0.1938). Actuarial allograft survival was similar between groups at 12 months (76% vs. 93% vs. 85%, NXM vs. FCXM+ vs. CDC+) and 24 months (76% vs. 89% vs. 85%, NXM vs. FCXM+ vs. CDC+) (P=0.0738). CDC+ allografts had a significant increase in early rejection episodes compared with NXM (46% vs. 7%, CDC+ vs. NXM) (P=0.003) or FCXM+ allografts (46% vs. 10%, CDC+ vs. FCXM+) (P=0.006). CDC+ allografts experienced significantly more rejection episodes per year than NXM (53% vs. 20%, CDC+ vs. NXM) (P=0.015) or FCXM+ allografts (53% vs. 23%, CDC+ vs. FCXM+) (P=0.02). CDC+ allografts had a significant increase in numbers of additional nonconventional therapeutic interventions compared to NXM allografts (0.9+/-0.5 vs. 0.2+/-0.1, CDC+ vs. NXM) (P=0.039). The presence of cytotoxic antibodies pretransplantation is associated with increased incidences of early rejection, and rejection episodes per year. With careful monitoring and aggressive therapeutic interventions the presence of cytotoxic antibodies are not deleterious to patient or liver allograft survival.

Disproportionate HLA matching may contribute to racial disparity in patient survival following cardiac transplantation.

The purpose of this study is to investigate the impact of recipient race as well as HLA matching upon long-term survival following heart transplantation (HTx). The study also determines whether the degree of HLA matching between Caucasians and African Americans differs. This study was a retrospective analysis of 336 males (77% Caucasians and 23% African Americans) transplanted between 1983 and 1994, all having received cyclosporine-based immunosuppression. The results showed African Americans were transplanted at a significantly younger age than Caucasians (39.1 +/- 11.2 vs. 48.2 +/- 10 yr). The composition of end stage cardiac disease was startlingly different. Caucasians demonstrated 62% CAD, 31% IDCM and 7% other, whereas African Americans exhibited 24% CAD, 69% IDCM and 7% other. The ten-year survival rate for African American male recipients was inferior to Caucasian males. Survival at 1, 3, 5, 7 and 9 yr was 83%, 73%, 63%, 51%, 46% for Caucasians and 70%, 58%, 51%, 38% and 32% for African Americans. Furthermore, African Americans received more poorly matched organs compared to Caucasians. Among Caucasians for Class I, 46%, 53% and 1% received poor, moderate and well matched hearts, respectively. In contrast, African American were allocated 65%, 33% and 0% of poor, moderate, or well matched hearts. An analysis of Class II data revealed the same pattern (63%, 35% 2% of Caucasians and 78%, 18%, and 4% of African Americans received poor, moderate or well matched hearts). Survival for Caucasians improved when moderately versus poorly matched for Class I. Class II matching did not have an effect. African Americans showed a similar trend, although statistical significance was not reached. When comparing equivalent degrees of matching, African Americans had inferior survival rate when poorly matched for Class I relative to Caucasians. No statistical difference was observed for moderate matched Class I or for Class II analysis.

The paradox of cytokine monitoring-predictor of immunologic activity as well as immunologic silence following cardiac transplantation.

The utility of cytokine monitoring to predict the onset of significant rejection was evaluated in 34 patients following heart transplantation. Serial blood levels of 5 cytokines involved in inflammation and immune activation(IL-1, IL-2, IL-6, IL-8, and TNF) were correlated with clinical outcome and endomyocardial biopsy scores. The majority of patients (68%) experienced a significant rejection during the study period. IL-6 and IL-8 levels were effective markers of significant rejection 2-4 days before diagnosis with EMBX. IL-6 and IL-8 levels of 15 and 1000 pg/ml predicted the onset of rejection with sensitivities of 75% and 66% and specificities of 86% and 76%, respectively. In contrast, IL-6 and IL-8 levels less than 15 and 400 pg/ml predicted a rejection-free course with sensitivities of 91% and 91% and specificities of 81% and 68%, respectively. The remaining cytokines differentiated patients experiencing a clinically unremarkable course from those experiencing mild-to-moderate rejection but did not discriminate rejection severity. IL-6 levels identified steroid and OKT3 resistance within 48 hr of antirejection therapy. IL-6 levels elevated to 197 +/- 20 pg/ml among steroid-resistant patients and normalized to 20 +/- 5 pg/ml among responders. IL-8 levels delineated OKT3 resistance. IL-8 levels rose to 3496 +/- 500 pg/ml among nonresponders, whereas levels fell to 152 +/- 50 pg/ml among responders. This study demonstrates that IL-6 and IL-8 are useful markers of rejection and therapeutic efficacy following heart transplation.

Positive pretransplant crossmatches predict early graft loss in liver allograft recipients.

We evaluated the influence of donor-recipient HLA compatibility and recipient pretransplant antidonor sensitization on liver allograft recipient survival. The overall graft survival results for 67 cyclosporine-prednisone treated liver allograft recipients at 3, 6, and 12 months posttransplant were 86%, 83%, and 83%, respectively. No significant differences were observed when comparing the one-year survivals of 81% vs. 85% for men and women or 80% vs. 85% for adult and pediatric patients. Similarly, no differences were observed when comparing one-year graft survivals for well vs. poorly matched recipients of 77% vs. 83% for recipients with < or = 2 HLA A, B vs. > 2 HLA A, B mismatches (MMs) and 82% vs. 82% for recipients with 0-1 HLA-DR MMs vs. 2 HLA-DR MMs, respectively. Pretransplant transfusion history and race also did not influence survival. Standard NIH (long-incubation) and anti-human globulin (AHG) crossmatches were performed. The 12% of recipients (8/67) displaying a positive NIH crossmatch experienced significantly poorer 3-, 6-, and 12-month survivals of 62% vs. 89%, 62% vs. 86%, and 62% vs. 86% (all P < 0.05), respectively, than the 59 NIH-crossmatch negative recipients. Similarly, the 16% (11/67) of recipients displaying a positive AHG crossmatch had significantly poorer 3-, 6-, and 12-month survivals of 63% vs. 91%, 54% vs. 89%, and 54% vs. 89% (all P < 0.05) respectively, than the 56 AHG crossmatch-negative recipients. NIH and AHG crossmatch-positive sera were treated with dithioerythritol (DTE) to establish whether reactivity was due to IgM or IgG immunoglobulin. One-year graft survivals of 65% vs. 30% (P < 0.05) were observed when the crossmatch-positive sera reactivities were due to IgM vs. IgG immunoglobulin. While graft survivals were improved when positive crossmatch serum reactivity was due to IgM, these survivals were still significantly poorer than when the crossmatches were completely negative (86% vs. 60%, P < 0.05 for NIH-negative vs. NIH-positive, but DTE-negative, and 88% vs. 77%, P < 0.05 for AHG-negative vs. AHG-positive, but DTE-negative). Therefore, an NIH- or AHG-positive crossmatch, due either to IgM or IgG reactivity, results in poor early (3- and 6-months) liver allograft survival. Crossmatch-positive recipients experienced significantly (P < 0.05) more rejections and more steroid-resistant rejections (P < 0.05) than crossmatch-negative recipients.(ABSTRACT TRUNCATED AT 400 WORDS)

Influence of HLA matching on rejections and short- and long-term primary cadaveric allograft survival.

Distribution of cadaveric donor kidneys, based upon the donor-recipient HLA match grade, remains one of the major controversies in transplantation. To determine whether matching results in fewer rejection episodes and better graft survival, we retrospectively studied our single-center patient population of 683 cyclosporine-prednisone-treated primary cadaveric renal allograft recipients. For 237 recipients of well-matched HLA A, B kidneys (< or = 2 HLA A, B mismatches [MM]) the 1-, 3-, 5-, and 7-year graft survivals of 76%, 66%, 62%, and 61%, respectively, were not significantly different from those of 71%, 65%, 63%, and 63%, respectively, for the 446 poorly matched HLA A, B (> 2 HLA A, B MM) recipients. Similarly, the 1-, 3-, 5-, and 7-year graft survivals for the 307 recipients of well-matched HLA-DR kidneys (0 or 1 DR MM) of 74%, 65%, 63%, and 61%, respectively, were not significantly different from those of 72%, 65%, 63%, and 62%, respectively, for the 366 poorly matched (2 DR MM) recipients. Patient survivals were comparable at each time point for well- vs. poorly matched recipients. Similarly, donor-recipient HLA A, B, and DR matching was not beneficial in retransplant recipients who were transplanted following negative NIH and antiglobulin (AHG) crossmatches when testing both historical (high-PRA) and pretransplant sera. Since rejection episodes may be a more sensitive indicator of immune response than graft loss, we also analyzed the relationship between donor-recipient HLA match grade and posttransplant rejections. A total of 60% (n = 413) of recipients experienced no rejections and had 1-, 3-, 5-, and 7-year graft survivals of 82%, 78%, 74%, and 73%, respectively; 32% (n = 215) of patients who experienced 1 rejection had 1-, 3-, 5-, and 7-year graft survivals of 58%, 48%, 44%, and 43%, respectively (P < 0.001 for graft survival of 0 vs. 1 rejection). The remaining 8% (n = 55) of recipients experienced more than 1 (> 1) rejection and had 1-, 3-, 5-, and 7-year graft survivals of 62%, 38%, 36%, and 36%, respectively (P < 0.001 for graft survival of 0 vs. > 1 rejection and P < 0.01 for graft survival of 1 vs. > 1 rejection). The mean numbers of rejections/patient experienced by well-matched vs. poorly matched recipients were comparable and not significantly different.(ABSTRACT TRUNCATED AT 400 WORDS)

Cyclosporine and rapamycin affect protein kinase C induction of the intracellular activation signal, activator of DNA replication.

Immunosuppressive agents may initiate their pharmacologic action by disrupting phosphorylation cascades critical to T lymphocyte intracellular signaling after alloantigen recognition. Activator of DNA Replication (ADR), a transduction signal sensitive to CsA as well as rapamycin (RAPA) immunosuppression, seemed a likely candidate for phosphoregulation. This communication reports the development of a cell-free assay wherein CsA and RAPA inhibit ADR induction by protein kinase C (PKC). ADR and PKC are inactive in the cytosol of resting cells. Endogenous PKC activity in quiescent lymphocytes was triggered with the tumor promoter PMA, leading to the appearance of ADR, an event subsequently quantitated by the ability of ADR to trigger [3H]thymidine triphosphate incorporation into isolated nuclei. After PKC induction, ADR activity is increased (890 +/- 120 vs. 3910 +/- 345 cpm, P < 0.001). In the presence of the PKC inhibitor H7, ADR activity fails to increase (100 +/- 50 vs. 3910 +/- 345 cpm, P < 0.001). The high levels of ADR found in PHA-stimulated cells is marginally affected by in situ PKC induction, although the sustained impact of H7 throughout the cell cycle suggests that ADR is constantly being phosphorylated. Titration of CsA or RAPA into the cell-free system inhibited ADR induction in a dose-dependent fashion. The utility of ADR induction as a marker for immunosuppression was investigated by comparing ADR inducibility in resting cells that had been preloaded with CsA or RAPA with the proliferative index of cells cultivated with CsA or RAPA. ADR induction directly correlated with CsA or RAPA proliferative inhibition. The results suggested that ADR is a constituent of the PKC phosphoregulatory cascade essential for cell cycle progression. The correlation between cell-free ADR inducibility and proliferative inhibition by CsA or RAPA suggest that this procedure may be useful for in vitro prediction of allografted patient immunologic response.

Influence of race on crossmatch outcome and recipient eligibility for transplantation.

Data from the Office of the Inspector General and the United Network for Organ Sharing suggested that black end-stage renal disease patients had unequal access to organ transplantation, in that they waited twice as long as white ESRD patients for a renal transplant. We hypothesized that preTx histocompatibility factors were more influential in determining how quickly an ESRD patient was transplanted than had been realized by either the Office of the Inspector General or UNOS. To test this hypothesis we compared the crossmatch reactivity of 378 white and 227 black ESRD patients awaiting a primary renal Tx against 100 consecutive cadaveric donors (80 white, 10 black, and 10 Mexican-American). A positive XM frequency of 16% (179 positive XM/1121 total XM) was observed when white ESRD patients were crossmatched against white donors. A comparable frequency of 21% (39/186) (+) XM was observed when white ESRD patients were crossmatched against black donors. Similarly, black ESRD patients crossmatched against black donors showed a 17% (20/115) (+) XM frequency. In contrast, a significantly higher (+) XM frequency of 43% (283/655) was observed when black ESRD patients were crossmatched against white donors (P less than 0.001). When black ESRD patients with a (+) preTx blood transfusion history were crossmatched against white donors, a 55% (241/438) (+) XM frequency was observed. This (+) XM frequency was 3.2 times greater than when black ESRD patients were matched against black donors (P less than 0.001). However, when untransfused black ESRD patients were matched against white donors only a 19% (42/217) (+) XM frequency was observed. Finally, preoperatively transfused black ESRD patients displayed a higher PRA of 47 +/- 12% vs. 27 +/- 10%, P less than 0.01, and a longer median waiting time to transplant of 329 vs. 181 days, P less than 0.01, than comparable white patients. The data suggest that presentization of blacks, following preTx blood transfusions from nonblack donors, predisposes black ESRD patients to present as immunologically inappropriate recipients for predominantly white donor allografts and results in their spending a longer time on renal transplant waiting lists.

AHG and DTE/AHG procedure identification of crossmatch-appropriate donor-recipient pairings that result in improved graft survival.

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.

Failure of prolyl-peptidyl isomerase to mediate cyclosporine suppression of intracellular activation signal generation.

Cyclosporine blocks the generation of a cytoplasmic activation protein, activator of DNA replication (ADR). The recent demonstration that cyclophilin is a CsA-sensitive prolyl-peptidyl-isomerase (PPIase), has prompted speculation that CsA immunosuppression is mediated by PPIase interaction with activation signals like ADR. We report that PPIase converts ADR from an inactive to an active form, but the interaction is resistant to CsA. ADR is a sensitive marker of CsA immunosuppression. ADR, extracted from the cytoplasm of PBLs stimulated with PHA, is not detectable in the cytoplasm of resting cells. ADR is quantitated by measuring uptake of 3H/thymidine triphosphate (3H/TPP) into isolated nuclei as a measure of DNA synthesis. The CsA-induced reduction of ADR content mirrored CsA-induced proliferative inhibition in intact cells. CsA concentrations of 1.5, 3, or 4.5 mM reduced T cell proliferation by 26%, 47%, and 58%, and ADR content by 32%, 45%, and 53%, respectively. The ability of PPIase to catalyze the transition of ADR between active and inactive forms was determined by measuring changes in DNA synthesis when 1 microgram/ml PPIase was added to (1) isolated nuclei, (2) nuclei plus ADR, and (3) nuclei plus the cytoplasmic fraction from resting cells. DNA synthesis in isolated nuclei (899 +/- 45 cpm) was unchanged by PPIase (1009 +/- 221 cpm). Addition of PPIase to ADR from activated cells marginally reduced ADR's capacity to trigger 3HTTP incorporation into isolated nuclei (ADR, 4113 +/- 106 cpm; PPIase-treated ADR, 3198 +/- 453 cpm), showing that PPIase cannot reduce ADR activity. However, treatment of resting cell cytoplasm with PPIase increased ADR activity 5-fold (899 +/- 46 vs. 5035 +/- 75 cpm). Addition of 1.5 or 3 mM CsA to resting cytoplasm, primed with PPIase (5035 +/- 75 cpm), resulted in 3HTPP incorporation of 6575 +/- 152 and 5076 +/- 168 cpm, respectively. Thus, PPIase activation of ADR is CsA-resistant. Furthermore, PPIase could not reverse CsA-induced inhibition of ADR. CsA (3 mM) treatment of PHA-stimulated cells rendered proliferation by 30% and ADR by 35%. ADR isolated from cells treated with CsA (9110 +/- 750 cpm) was not increased by treatment with PPIase (9185 +/- 449 cpm). These findings suggest that PPIase converts ADR from an inactive to an active form. However, the mechanism of CsA inhibition of ADR is neither mediated nor overridden by PPIase.