Altered response to pneumococcal polysaccharide in offspring of immunologically paralyzed mice.

The dose of pneumococcal polysaccharide type III required to induce immunologic paralysis in newborn offspring of immunologically paralyzed mice was one-tenth of the corresponding paralyzing dose for newborn offspring of normal mice. Similarly, immunization of the offsprinig of the paralyzed mice was accomplished with one-tenth the dose of polysaccharide necessary to immunize normal newborn mice. The altered susceptibility of newborn mice from paralyzed mothers to the induction of both paralysis and immunity was predicted from theories of antibody formation which postulate that the induction of tolerance or immunity is controlled by the concentration of natural antibodies specific for the antigen used.

Impact of rituximab therapy for treatment of acute humoral rejection.

INTRODUCTION: Antibody mediated rejection (AMR) is associated with a greater incidence of allograft loss because traditional approaches - pulse steroid or anti-lymphocyte antibodies are usually ineffective. This retrospective analysis documented the benefit of rituximab administration in addition to plasmapheresis (PP). METHODS: We retrospectively reviewed the data from 54 kidney transplant patients treated for AMR between 2001 and 2006, including 26 patients who received PP plus rituximab (Group A), versus 28 subjects who underwent PP without rituximab (Group B). Only patients whose serum IgG levels were below normal values received intravenous gamma globulin (IVIG). In addition to clinical and demographic variables we evaluated graft/patient survivals at two years post-diagnosis, Banff classification of rejections, serum creatinine and calculated GFR values at baseline, rejection, resolution as well as three, six, 12 and 24 months thereafter. RESULTS: The demographic features of the cohorts showed no significant differences. The two-year graft survival for patients treated with rituximab plus PP was 90%, significantly better than 60% in the PP cohort (p = 0.005). Upon multivariate analysis administration of rituximab was the most significant factor (>or= 0.009); whereas, IVIG also produced a useful effect (p = 0.05). Neither the mean (>or= 0.42) nor the slope (p = 0.25) of GFR values showed a significant difference among salvaged kidneys over 24 months after completion of AMR treatment. The rates and types of infectious complications at three and six months did not show significant differences or impact on graft survival. CONCLUSION: Addition of rituximab improved the outcomes of PP treatment of antibody mediated rejection episodes.

Selective corticosteroid and calcineurin-inhibitor withdrawal after pancreas-kidney transplantation utilizing thymoglobulin induction and sirolimus maintenance therapy.

Of 25 simultaneous pancreas-kidney transplant (SPK) recipients treated with thymoglobulin induction, sirolimus and reduced-dose cyclosporine (CsA), 18 low-immune responders (non-African-Americans, PRA < 30%) were withdrawn from prednisone on post-transplant day 5, whereas seven high-immune responders continued on prednisone. Most high- and low-immune responder recipients were converted from CsA to mycophenolic acid (MPA) at six months post-transplantation. At a mean follow-up of 28 +/- 10 months, two pancreas grafts were lost to pancreatitis. There were no patient or kidney graft losses, but one acute rejection episode. At 28 +/- 11 months, all 18 low-responder recipients remain steroid-free. Twenty recipients (14 low and six high-immune responders) were converted from CsA to MPA. During conversion, immune response was monitored by Flow-PRA and T-cell stimulation (Cylex) assays. Nineteen of 20 recipients displayed a post-conversion PRA of 0%, whereas one highly sensitized patient expressed a post-conversion PRA of 67%. Fifty-eight percent of individual T-cell stimulation scores were in the hypo-responsive range. Twelve of 18 low-immune responders are both steroid and CsA-free at a mean follow-up of 17 +/- 13 months, whereas five of seven high-immune responders remain CsA-free at a mean follow-up of 11 +/- 10 months. These data suggest that thymoglobulin induction with combined sirolimus and CsA maintenance therapy permits immunosuppression minimization in selected SPK recipients.

Active T-rosette-forming cells in the peripheral blood of cancer patients.

We studied a subpopulation of the thymus-dependent rosette-forming lymphocytes from the peripheral blood of normal individuals and of untreated patients with solid tumors or hematological cancers. This subpopulation of the thymus-dependent rosette-forming cells (T-RFC), termed the "active T-RFC," may be relatively more immunocompetent than the total thymus-dependent population. The mean percentages and absolute numbers of active T-RFC of 40 healthy adult controls were 25.8 +/- 4.3 and 626 +/- 213, respectively. There was no difference in the percentage of active T-RFC between the controls (smokers and nonsmokers) and the 102 untreated patients with solid (localized or metastasized) tumors, 4 patients with Hodgkin's disease, or the 10 patients with non-Hodgkin's lymphomas. However, the absolute number of active T-RFC was significantly less in the cancer patients than in the controls. Eight patients with chronic lymphocytic leukemia had lower percentages but higher absolute numbers of active T-RFC, whereas 6 patients with multiple myeloma had higher percentage and lower absolute numbers than the controls. Following radiation therapy, 61 patients with solid tumors showed no difference in the percentage of active T-RFC, but the corresponding absolute numbers declined significantly. A good correlation was seen with patients having positive microbial skin test responses and normal percentage of active T-RFC. The significance of both the percentages and absolute numbers of active T-RFC and their relationship to patient status are discussed.

A pilot study of immunosuppression minimization after pancreas-kidney transplantation utilizing thymoglobulin induction and sirolimus maintenance therapy.

AIMS: To determine outcomes utilizing thymoglobulin and sirolimus immunosuppression, with early steroid withdrawal in low-immune responder pancreas-kidney (SPK) recipients, and conversion from cyclosporine (CsA) to mycophenolic acid (MPA) in all recipients at 6 months posttransplantation. METHODS: SPK recipients received thymoglobulin, sirolimus, and reduced-dose CsA immunosuppression. Low immune responders (non-African-Americans with a pretransplant PRA < 30%) were withdrawn from prednisone on posttransplant day 5 and high immune responders were continued on prednisone. All recipients were converted from CsA to MPA at 6 months posttransplantation. During conversion, recipient immune response was monitored by flow PRA and a T-cell stimulation assay (Cylex). RESULTS: With a mean follow-up of 9 +/- 4 months, one pancreas was lost to pancreatitis, with no patient or kidney losses and no acute rejection episodes. All eight low immune responder patients were steroid-free at 9 +/- 5 months posttransplantation. Seven patients (five low and two high immune responders) with at least 6-month follow-up were converted from CsA to MPA. One high immune responder with a pretransplant PRA of 43% remained with a PRA of 53% +/- 2% postconversion. The second high immune responder had a pretransplant PRA of 34% and a postconversion PRA of 0%. The five low immune responders had a mean pretransplant PRA of 16% +/- 15% and a postconversion PRA of 0% (P < .01). The Cylex assay resulted in 67% low responsiveness for both high and low immune responders. CONCLUSION: Thymoglobulin induction with sirolimus maintenance therapy permitted immunosuppression minimization in selected pancreas transplant recipients. Posttransplant evaluation revealed a diminished (regulated) immune response in six of seven patients.

Graft survival and immune regulation of pancreas allograft recipients induced with thymoglobulin, sirolimus, and cyclosporine.

BACKGROUND: Our objective was to determine the impact of thymoglobulin-sirolimus-cyclosporine immunosuppression on the alloimmune response of pancreas-kidney transplant recipients. METHODS: Thirty-six pancreas transplant recipients received an induction protocol of thymoglobulin, sirolimus, reduced-dose cyclosporine, and corticosteroids. A subset of 10 recipients were also enrolled in a study to measure immune responsiveness. Flow PRA-determined HLA antibody, donor-specific flow cytometry crossmatching (FCXM), T-cell subset, and suppressor cell assays were performed at various timepoints during the first posttransplant year. RESULTS: One-year patient, kidney, and pancreas survivals were 97%, 94%, and 92%, respectively. There was 1 death due to sepsis, and 1 kidney and 2 pancreas graft losses. There were no acute rejection episodes. Recipients in the immune-monitoring study displayed depression of CD3, CD4, and CD8 counts (<80%) until 3 months posttransplant. At transplantation, 9 of 10 patients displayed <10% class I HLA antibody. By 3 months, 7 of 10 showed a transient elevation in class I HLA antibodies, with 2 patients expressing >80% flow PRA. At transplant 1 patient was FCXM-positive, whereas, by 3 months posttransplant, 2 of 10 patients demonstrated a positive FCXM. There were no clinical consequences to either the presence of HLA antibody or the positive FCXMs. By 6 months, 7 of 9 patients expressed immunoregulatory suppressor cell activity. CONCLUSIONS: The absence of acute rejection events was likely due to inhibition of donor-specific immunity. Seventy percent of patients demonstrated an early non-donor-directed HLA antibody response that had no adverse effect on graft function and 78% of the patients displayed immunoregulatory suppressor cell function, probably contributing to the successful clinical outcome.

Transplantation of single pediatric kidneys into adult recipients.

AIM: To evaluate the outcome of single pediatric kidneys transplanted into adult recipients. METHODS: A retrospective single-center review was performed of transplants from donors less than 5 years of age. Outcomes were compared with recipients of grafts from donors 18 to 45 years transplanted during the same time period. RESULTS: Thirty single renal transplants from pediatric donors and 117 transplants from adult donors between 18 and 45 years of age were performed during the study period. The mean age of the pediatric donors was 2.9 +/- 0.8 years versus 31.5 +/- 8.9 years for adult donors (P < .001). The mean age of the recipients of pediatric donors was 41.9 +/- 13 years versus 48 +/- 12.6 years for recipients of adult grafts (P = .020). The mean recipient weight of pediatric donors was 55.9 +/- 7.8 kg versus 78.0 +/- 17.7 kg for recipients of adult donors (P < .001). Sixty-six percent of pediatric donor recipients were of female gender compared to only 36% of adult donor recipients (P = .005). Death-censored actuarial graft survivals at 1 and 4 years for recipients of pediatric donor grafts were 90% and 85% compared to 93% and 85% for recipients of adult donor grafts (P = NS). The mean calculated creatinine clearances of adult donor graft recipients at 1 and 4 years posttransplantation were 70.8 +/- 26.5 and 73.7 +/- 27.2 mL/min, respectively, compared to 50.3 +/- 20.1 and 56.3 +/- 21.4 mL/min for pediatric donor grafts (P < .01 at 1 and 4 years). CONCLUSION: The use of single pediatric donor kidneys provides an excellent opportunity to safely expand the donor pool.

Amyotrophic lateral sclerosis. Associated clinical disorders and immunological evaluations.

We examined the family history and associated diseases in 58 patients with amyotrophic lateral sclerosis (ALS), as well as the T-cell phenotypes and functions in 46 consecutive patients with this disorder. A family history of thyroid disease was present in 19%, and an additional 21% of patients described family members with other possible autoimmune disorders. In 19% of the patients with ALS either past or present thyroid disease was documented. Eleven of 47 additional patients with ALS had significant elevations of microsomal and/or thyroglobulin antibody levels. The T-cell phenotypes and functions were comparable in the ALS and control groups, with the exception of the presence of Ia antigen. In patients with ALS, 11.9% of the T cells were positive for the la antigen, while in both a normal control population and a non-ALS neurologic disease population, only 6.4% of T cells have this antigenic determinant. These data support involvement of autoimmune mechanisms in ALS.

Immune studies in human immunodeficiency virus infection with myasthenia gravis: a case report.

A patient with human immunodeficiency virus infection had cellular and humoral immune responses studied longitudinally from the onset of generalized myasthenia gravis. Progressive decline in CD4+, CD45R+ and CD4+, CDw29+ T-cells, cellular immune responses to alloantigen and mitogen stimulation, and acetylcholine receptor antibody titers were associated with clinical improvement of all myasthenic symptoms.

Delayed hypersensitivity in the hamster cheek pouch.

A method is described for eliciting a delayed hypersensitive cell mediated immune response in the Syrian hamster cheek pouch. Using a sensitizing dose of 0.1% DNCB and a challenge dose of 0.01% DNCB 14 days later resulted in a histologically positive lymphocyte infiltrate in the challenge area. These results indicate that the hamster cheek pouch may not be an immunologically inert site and may be used for cell mediated immunity studies.

A modified assay for the detection of human adult active rosette forming lymphocytes.

A modified procedure is described for the detection of a sub-population of peripheral blood T-cells in normal human adults. This sub-population of T-cells has been called the active rosette forming cell by others. The advantages of this modified procedure compared with that previously described are: 1) the elimination of 60 mon incubation period for lymphocytes prior to the assay and 2) the elimination of the need for fetal calf serum in the assay. The mean percentage of RFC in the peripheral blood of 80 healthy adult controls using the modified procedure is 25.2+/-5.5% in buffer lacking FCS. The results were highly reproducible and comparable to the results obtained using the procedure previously described. It was concluded that a 60 min pre-incubation of lymphocytes and the use of FCS are not necessary when the appropriate SRBS: lymphocyte ratio is used in the determination of human adult active RFC. The use of this procedure in evaluating the immune competence of cancer patients is discussed.

Effect of cyclosporine on rubella virus-specific immune responses in chronic progressive multiple sclerosis.

Cyclosporine A (CsA) has been used in putative autoimmune diseases after sensitization to unknown antigens. We have previously shown that CsA prevented continued activation of T-cells in chronic progressive multiple sclerosis (CPMS) patients. The current study was undertaken to determine whether CsA, or CsA and prednisone (CsA + P) could suppress immune responses to a common recall antigen. Serum antibody levels were higher in all CPMS patients than age-matched normal controls. However, rubella antibody titers in the CsA or CsA + P groups were no different from a placebo-treated CPMS patient group. The lymphocyte responses to inactivated rubella virus of CsA and CsA + P-treated CPMS patients were lower than placebo and control but not statistically different. Therapy with both CSA and CSA + P was associated with significantly lower panel mixed leukocyte responses and Ta1 expression than in the placebo-treated group; CD3, CD4, CD8 antigen expression and active rosette formation by T-cells were similar for the three CPMS groups. These results suggest that while CsA exerts measurable effects on non-specific indicators of cellular immunity in CPMS patients, it may not be as effective in suppressing pre-existent specific immune responses.

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.

Serologic methods for the early diagnosis of Pneumocystis carinii infection in renal allograft recipients.

Because of the nephrotoxic action of trimethoprimsulfamethoxazole (TMP-SMX) in cyclosporine (CsA)-treated patients, combined with the (CsA)-treated patients, combined with the possibility of selecting resistant gram-negative or Nocardia asteroides organisms, a monitoring tool to detect early Pneumocystis carinii (PC) infection permitting a selective treatment approach is highly desirable. A review of 401 consecutive renal transplants revealed 26 cases (18 suspected and 8 histologically proved) of PC infection in 21 cadaver and 5 living-related renal recipients. The diagnosis was confirmed in 8/18 patients who were invasively studied by open-lung biopsy (1/2), bronchoscopy with transbronchial biopsy (4/9), bronchoscopy with brushing (1/2), bronchoscopy with bronchoalveolar lavage (2/5), and transpleural needle biopsy (0/1)-yielding a confirmed incidence of 2% (8/401). All positive invasive studies had been performed prior to or within 24 hr of the inception of TMP-SMX therapy. Nine of ten negative invasive studies were performed after more than 24 hr of treatment. The mean time from transplantation to the onset of clinical symptoms was 2.5 +/- 1.5 months. The infection rate would be 6.5%, assuming all 18 suspected cases would be PC-positive if studied pretreatment. In order to assess the efficacy of a variety of serologic methods of PC detection, qualitative counter-immunoelectrophoresis (CIE) for P carinii antigen (PC-Ag), IgG antibody reactive with PC (enzyme-linked immunosorbent assay [ELISA]), and a latex particle agglutination test (LPA) were performed on 279 sera; 85 sera from the 26 suspected or proved cases, 100 sera from normal age-matched controls, and 94 sera from 78 asymptomatic allograft recipients followed as outpatients. In the eight histologically proven cases, CIE was positive in only 3/8 and turned positive late in the clinical course. LPA was positive in all histologically proved cases; however, it was also positive in 60% of asymptomatic renal recipients. In cases that developed clinical disease, LPA increased in titer weeks to months prior to the onset of symptoms. Additionally, LPA titers decreased or stabilized during successful TMP-SMX therapy, providing an early therapeutic index. Measurement of anti-PC IgG was not useful per se, as it was elevated in both controls and documented PC infection. The combination of very low antibody titer (less than or equal to 1:16) with a positive or increasing LPA PC-Ag titer appeared to be disease-predictive.(ABSTRACT TRUNCATED AT 400 WORDS)

Oral administration of human or murine interferon alpha suppresses relapses and modifies adoptive transfer in experimental autoimmune encephalomyelitis.

Chronic relapsing experimental autoimmune encephalitis (CR-EAE) is an inflammatory process of the central nervous system (CNS) that closely resembles the human disease multiple sclerosis (MS). EAE was induced in SJL/J mice and following recovery from the initial attack, animals were fed varying doses of human or murine interferon alpha (IFN-alpha), or mock IFN three times per week. After relapse, concanavalin A-activated spleen cells were transferred adoptively from orally fed animals into recipient animals. Oral administration of human or murine IFN-alpha suppressed relapse in actively immunized animals, modified adoptive transfer of EAE, and decreased mitogen/antigen proliferation and IFN-gamma secretion in both donors and recipients. IFN-alpha acts orally by modifying the encephalitogenicity of donor spleen T cells.

Serial immune evaluation of cyclosporine- and placebo-treated multiple sclerosis patients.

During an ongoing clinical trial of cyclosporine (CsA) immunosuppression therapy for chronic progressive multiple sclerosis (MS), a comparison was made of the immune responses of 18 CsA- and 18 placebo (P)-treated MS patients. Patients randomized to receive either CsA or P had identical entry immune profiles. However, these MS patients displayed significantly increased T-helper:T-suppressor (TH:TS) ratios (P less than 0.01), percentage (%) active-T (P less than 0.01), % Ia+-T (P less than 0.05) and % Ta1+-T (P less than 0.01) cell phenotypes when compared to age-matched normal controls. Further, the MS-P-treated patients displayed significant increases (all P less than 0.01) in % pan-T, % helper-T, % active-T and % Ta1+-T cell phenotypes as well as panel mixed lymphocyte culture (panel MLC) functional responsiveness from entry to cumulative 12-month study data. In contrast, the MS-CsA-treated patients only displayed an increased % pan-T cell phenotype. The cumulative 12-month follow-up data showed that the MS-P-treated patients displayed significantly higher immune parameters than the MS-CsA-treated patients for % pan-T (P less than 0.05), % helper-T (P less than 0.01), TH:TS ratio (P less than 0.05), % active-T (P less than 0.01), % Ta1+-T cells (P less than 0.01) and panel MLC stimulation index (P less than 0.01). Thus, MS-CsA-treated patients did not display the progressive immune activation seen on serial evaluation during the follow-up time period that characterized the placebo-treated MS group.

Immunopharmacodynamic evaluation of cyclosporine-treated renal allograft recipients.

Since the mode of action of cyclosporine (CsA) in man is incompletely understood, there are no monitoring tools to assess immunosuppressive effect in vivo. In vitro CsA inhibits lymphoproliferation in response to allogeneic and mitogenic stimuli, presumably due to reversible suppression of T helper cell generation of interleukin-2. Therefore the present studies examined the immunosuppressive effect of patient sera on a third-party mixed lymphocyte reaction (MLR) as a pharmacodynamic approach to quantify patient responses to CsA administration. Four kinetic patterns of in vitro immunosuppressive activity were discerned: 24/28 (86%) patients showing two cycles of MLR inhibition--namely, a first peak corresponding to absorption of CsA and an independent second peak of immunosuppression (type I), were free of rejection; while 17/23 (74%) patients demonstrating one cycle corresponding to the peak of CsA absorption (type II) suffered rejection episodes (P less than 0.001). In addition, 20 patients generating continuously high levels of in vitro serum activity (type III) were almost all free of rejection, but manifested nephrotoxicity; while two patients showing continuously low levels (type IV) suffered graft loss due to irreversible rejection (P less than 0.01). Thus failure to display either a second peak or continuously high levels of MLR inhibition was associated with a markedly increased incidence of rejection (76% versus 16%). The in vitro functional characteristics of peak-2 were similar to those of CsA, as assessed by the kinetics of inhibition of MLR lymphoproliferation or cell-mediated lympholysis (CML), and by gross chemical properties of partitioning into organic solvents and heat stability. These findings suggest that pharmacodynamic analysis by MLR inhibition not only affords a useful parameter of immunosuppression, but also may provide an in vitro model to dissect the generation and biotransformation of active CsA metabolites.

Suppression of mixed leukocyte culture using leukocytes from normal individuals, uremic patients, and allograft recipients.

The suppressor cell function of peripheral blood mononuclear leukocytes (MNLs) from normal individuals, renal failure patients, and allograft recipients was evaluated using a suppressor cell assay wherein putative suppressors were added at the initiation of a third-party, one-way mixed leukocyte culture (MLC). MNLs from renal allograft recipients displayed the greatest suppressive activity (P less than 0.05): 50% suppression was achieved with 0.5 x 10(5) MNLs from allograft recipients, 2 x 10(5) MNLs from end stage renal disease patients, or 4 x 10(5) MNLs from normal individuals. The frequency of tests displaying positive MLC suppression using 1 x 10(5) MNLs was 90% (383 of 426) for allograft recipients compared with 60% (83 of 138) for renal failure patients and 28.2% (29 of 103) for normal individuals (P less than 0.01). The suppression displayed by 1 x 10(5) MNLs from potential allograft recipients receiving more than five pretransplant blood transfusions (BTs) was equal to that of 4 x 10(5) MNLS from patients receiving less than five BTs (P less than 0.05). Moreover, the frequency of positive MLC suppression from patients treated with more than five versus less than five BTs was 80% versus 40% (P less than 0.001). Although suppression of MLC appeared to be mainly dependent on an esterase (+)-adherent cell, there was no significant difference between the percentage of esterase (+) cells in unfractionated MNLs from the three groups of individuals, suggesting that MLC suppression was attributable to the functional performance rather than the number of suppressor MNLs. Preliminary data suggest a relationship between the level of MLC suppressive activity detected pretransplant and the outcome of the allograft at 6 months.

Production of synergistic but nonidentical mechanisms of immunosuppression by rapamycin and cyclosporine.

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.

Sensitivity of intracellular signals responsible for cell cycle progression to cyclosporine.

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.