Tudor Birsan

Quantification of immunosuppression by flow cytometry in stable renal transplant recipients.

The current standard of monitoring transplant patients by drug levels is not optimal because it does not take into account the different and individual effects of immunosuppressive drugs on each patient. In this study, the authors tested immune function assays for monitoring transplant patients. Blood was collected from stable renal transplant patients treated with cyclosporin, mycophenolate mofetil, and prednisone (n = 8), and from healthy volunteers (n = 12). Lymphocyte proliferation, expression of T-cell surface activation antigens (CD25, CD71, CD11a, CD95, CD154), production of intracellular cytokines (IL-2, INF&ggr;, TNF&agr;), and lymphocyte subsets (CD4, CD8, CD16, CD20) were assessed by flow cytometry. Lymphocyte proliferation, expression of T-cell surface activation antigens, and production of intracellular cytokines were significantly decreased in transplant recipients compared with healthy control volunteers. The combined effects of several immunosuppressive drugs in renal transplant recipients can be quantitated with immune function assays in whole blood. This new method may be helpful to achieve an optimal level of immunosuppression for each patient.

In vivo evaluation of the novel calcineurin inhibitor ISATX247 in Non-Human primates

BACKGROUND ISA(TX)247 is a novel calcineurin inhibitor that has shown more potency than cyclosporine in vitro. This is the first in vivo study of the effects of ISA(TX)247 on lymphocyte functions in non-human primates. METHODS Groups of cynomolgus monkeys were treated orally twice daily for 7 days, each dose consisting of 25 mg/kg cyclosporine (n = 5), 25 mg/kg ISA(TX)247 (n = 6) or 50 mg/kg ISA(TX)247 (n = 6). Levels of cyclosporine and ISA(TX)247 in whole blood were measured by liquid chromatography/mass spectrometry. After mitogen stimulation, lymphocyte proliferation was assessed by tritium-labeled thymidine incorporation and by flow cytometry (expression of proliferating cell nuclear antigen in cells in S/G(2)M phase). Flow cytometry was also used to assess production of intracellular cytokines by T cells (interleukin-2, interferon-gamma, tumor necrosis factor-alpha) and expression of T-cell surface activation antigens (CD25, CD71, CD11a, CD95, CD154). RESULTS Trough (C(14 hr)) and peak (C(3 hr)) drug levels, as well as area under the concentration-time curve, were significantly higher for cyclosporine than ISA(TX)247 (370 ng/ml vs 70 ng/ml, 877 ng/ml vs 303 ng/ml and 6,262 ng. h/ml vs 1,979 ng. h/ml, respectively). On Day 7 at C(14 hr), lymphocyte proliferation had been suppressed by approximately 50% in all groups compared with proliferation before treatment. Three hours after dosing, lymphocyte proliferation was inhibited significantly more by ISA(TX)247 (approximately 80%, with no differences between the two ISA(TX)247 dose levels) than by cyclosporine (65% inhibition). Similar differences between the immunosuppressive effects of ISA(TX)247 and cyclosporine were found for inhibition of expression of T-cell surface activation antigens. Despite lower ISA(TX)247 exposures compared with cyclosporine, the cyclosporine treatment only rarely suppressed cytokine production more than treatment with ISA(TX)247. CONCLUSIONS In non-human primates, ISA(TX)247 produces a greater or similar inhibition of lymphocyte proliferation, expression of T-cell activation surface antigens, and cytokine production when compared with cyclosporine, despite ISA(TX)247s lower blood levels and total exposure. We conclude that ISA(TX)247 suppresses diverse T-cell functions more potently than cyclosporine in non-human primates in vivo.

Sirolimus (Rapamycin) Monotherapy Prevents Graft Vascular Disease in Nonhuman Primate Recipients of Orthotopic Aortic Allografts

Background—Delayed treatment with sirolimus (SRL) halts progression of graft vascular disease (GVD) in nonhuman primate (NHP) aortic allograft recipients. In this study, we investigated whether SRL monotherapy prevents the development of GVD. Methods and Results—Pairs of 3-cm infrarenal aortic segments were exchanged between mixed lymphocyte reaction–mismatched, blood group–compatible NHPs (n=12). Six NHPs were untreated controls, and 6 were treated orally with SRL starting on the day of transplantation. Follow-up was 105 days. SRL doses were adjusted individually by assessing SRL blood concentrations, immune function, and clinical status. The severity of GVD was determined every 3 weeks by intravascular ultrasound, which quantified intimal area (IA) and intimal volume (IV) for the middle 1-cm graft segments. The mean±SEM SRL plasma levels were 14.5±9 ng/mL. In grafts from treated NHPs, IA and IV values on days 63, 84, and 105 were significantly lower than for controls (P <0.05 to P <0.001). On day 105, in the grafts from SRL-treated NHPs compared with grafts from controls, values (mean±SEM) were IA, 2.9±0.9 versus 5.5±0.7 mm2, P <0.001 and IV, 29.6±4.6 versus 55.2±2.8 mm3, P <0.001; IA and IV values for grafts from SRL-treated NHPs did not increase significantly between days 21 and 105. Conclusions—We show that SRL monotherapy prevented GVD in NHP aortic allograft recipients, suggesting the value of SRL for controlling GVD in clinical transplantation.

Comparison between C0 and C2 monitoring in de novo renal transplant recipients: retrospective analysis of a single-center experience.

Background. Monitoring immunosuppression with cyclosporine microemulsion formulation (CsA-MEF) by using 2-hour CsA blood levels (C2) has been strongly recommended after kidney transplantation. The aim of our study was to evaluate the impact of C2 monitoring on the clinical outcome early after transplantation in a single-center setting. Methods. Nonsensitized, consecutive, de novo cadaveric kidney-transplant recipients were treated with CsA-MEF, mycophenolate mofetil, and steroids. Patients receiving transplants after January 2002 (n=89) were prospectively monitored by C2 levels (target: 1,500±200 ng/mL [fluorescence-polarization immunoassay]). They were retrospectively compared with the patients receiving transplants during 2001 (n=88) who had been monitored by C0 levels (target: 250±50 ng/mL). Results. In the intention-to-treat analysis, 40 (45.4%) patients in the C0 group and 25 (28.1%) patients in the C2 group received treatment for rejection (P=0.017). The incidence of histologically verified rejection of Banff grade I or higher was 20.45% in the C0 group and 13.48% in the C2 group (P=0.235). In the per-protocol analysis, incidence of treated rejection was 24.7%, and incidence of histologically verified rejection of Banff grade I or higher was 12.35% in the C2 group (P=0.004 and 0.160, respectively, vs. C0). Mean CsA-MEF doses were 1.7 to 2 times higher in the C2 group than in the C0 group throughout follow-up (P=0.019). In the C2 group, target C2 levels were achieved on average 4 days after transplantation, and there was no significant difference in C2 levels between patients who rejected and patients who did not reject. Conclusion. Kidney-transplant recipients monitored by C2 levels receive significantly higher doses of CsA-MEF and have a lower incidence of early acute allograft rejection than patients monitored by C0 levels. In C2 monitored patients, C2 levels are not predictive for the incidence of early allograft rejection.

Case Report: Pneumatosis Intestinalis with Clostridium difficile Colitis as a Cause of Acute Abdomen After Lung Transplantation

Pneumatosis inte stinalis is a rare and, in the majority of cases, unexpected disorder de ® ned as accumulation of gas in the submucosa and/or the subse rosa of the large or small inte stine s. It is associated with a wide varie ty of diseases, and its pathoge nesis is still obscure . We report a case of acute abdome n with sepsis syndrome in a patient after lung transplantation in whom the diagnosis of pneumatosis inte stinalis coli was made by sonography and compute rtomography. To the best of our knowledge this is the ® rst case of pneumatosis inte stinalis in a lung transplant recipient associated with acute abdomen.

Treatment by Mycophenolate Mofetil of Advanced Graft Vascular Disease in Non‐Human Primate Recipients of Orthotopic Aortic Allografts

Failure to control chronic graft dysfunction [e.g. graft vascular disease (GVD)] is the primary cause of immunologic graft failure. This is the first study of mycophenolate mofetil (MMF) for the treatment of GVD in non‐human primate recipients of aortic allografts. Abdominal aortic allografts were exchanged between mixed leukocyte reaction (MLR) ‐mismatched, blood‐group‐compatible cynomolgus monkeys. Six control recipients were untreated. Individualized treatment with frequent dose adjustments of MMF insured that treatment was close to the maximum tolerated dose (mean 99.2 mg/kg/day). Immune‐mediated injury proceeded unhindered until day 45, after which MMF treatment began. Changes in intimal volume (IV) were quantified by intravascular ultrasound (IVUS) and compared to histology on day 105. Serial IVUS measurements of IV (mm3) in controls showed progressive GVD. In four out of six animals, MMF was well tolerated, thus enabling optimum treatment; in all these animals, IV was significantly less than in the control animals (p = 0.02). In the two remaining animals, high doses were not tolerated; at day 105, there was no significant difference in IV between them and the controls. We found a significant correlation between the mean MMF tolerated dose and the inhibition of progression of IV (r = −0.88, p = 0.015). When high MMF doses were tolerated, MMF slowed progression of GVD.

Lung Transplantation for Primary Pulmonary Hypertension and Giant Pulmonary Artery Aneurysm

We report the case of an 18-year-old patient with a giant pulmonary artery aneurysm and primary pulmonary hypertension who was successfully treated with bilateral lung transplantation and complete reconstruction of the pulmonary artery.

Immunosuppressive effects of surgery assessed by flow cytometry in nonhuman primates after nephrectomy

Despite previous studies suggesting that surgery cause immune suppression, the underlying biologic mechanisms have not been studied using advanced immune function assays. Unilateral nephrectomy was performed in nonhuman primates. Blood was collected before surgery and at different time‐points through 14 days after surgery. Lymphocyte proliferation (expression of proliferating cell nuclear antigen in cells in S/G2M‐phase), production of intracellular cytokines [interleukin (IL)‐2, interferon (IFN)‐γ, tumor necrosis factor (TNF)‐α] and expression of surface‐activation antigens (CD25, CD71) on T‐lymphocytes were assessed in whole blood using flow cytometry. Results were compared with nonoperated control animals. The procedure caused a decrease of 25% in absolute lymphocyte count on postoperative day 3. Inhibition of lymphocyte proliferation was maximal on postoperative day 1 (55% normalized to preoperative values) and was detectable until postoperative day 7, when it was 25%. Expression of T‐cell activation antigens was decreased during the first postoperative week with a maximum on postoperative day 1 for CD71 (29%) and on postoperative day 3 for CD25 (49%). Intracellular production of cytokines by T cells was decreased only on postoperative day 1 (50% for IL‐2, 29% for IFN‐γ and 22% for TNF‐α). Immune functions returned to presurgery values by day 14. A major surgical procedure severely inhibits lymphocyte proliferation and various T‐cell functions up to 1 week postoperatively.

Comparison Between C0 And C2 Monitoring In De Novo Renal Transplant Recipients

BACKGROUND Monitoring immunosuppression with cyclosporine microemulsion formulation (CsA-MEF) by using 2-hour CsA blood levels (C2) has been strongly recommended after kidney transplantation. The aim of our study was to evaluate the impact of C2 monitoring on the clinical outcome early after transplantation in a single-center setting. METHODS Nonsensitized, consecutive, de novo cadaveric kidney-transplant recipients were treated with CsA-MEF, mycophenolate mofetil, and steroids. Patients receiving transplants after January 2002 (n=89) were prospectively monitored by C2 levels (target: 1,500+/-200 ng/mL [fluorescence-polarization immunoassay]). They were retrospectively compared with the patients receiving transplants during 2001 (n=88) who had been monitored by C0 levels (target: 250+/-50 ng/mL). RESULTS In the intention-to-treat analysis, 40 (45.4%) patients in the C0 group and 25 (28.1%) patients in the C2 group received treatment for rejection (P=0.017). The incidence of histologically verified rejection of Banff grade I or higher was 20.45% in the C0 group and 13.48% in the C2 group (P=0.235). In the per-protocol analysis, incidence of treated rejection was 24.7%, and incidence of histologically verified rejection of Banff grade I or higher was 12.35% in the C2 group (P=0.004 and 0.160, respectively, vs. C0). Mean CsA-MEF doses were 1.7 to 2 times higher in the C2 group than in the C0 group throughout follow-up (P=0.019). In the C2 group, target C2 levels were achieved on average 4 days after transplantation, and there was no significant difference in C2 levels between patients who rejected and patients who did not reject. CONCLUSION Kidney-transplant recipients monitored by C2 levels receive significantly higher doses of CsA-MEF and have a lower incidence of early acute allograft rejection than patients monitored by C0 levels. In C2 monitored patients, C2 levels are not predictive for the incidence of early allograft rejection.

Einfluss von Mycophenolat Mofetil auf die Transplantat-Vaskulopathie nach allogener Aortentransplantation im Primaten-Modell

Failure to control chronic graft dysfunction (eg, graft vascular disease (GVD)) is the primary cause of graft failure. We have shown in rats that mycophenolic acid (MPA), the active metabolite of mycophenolate mofetil (MMF), inhibits: 1) smooth muscle proliferation in vitro, 2) intimal thickening in arterial and heart transplants and 3) neointima formation after balloon catheter injury in native arteries. We now report the first study of MMF for the treatment of GVD in nonhuman primates. MethodsAortic allografts were exchanged between MLR mismatched, blood group compatible cynomolgus monkeys. 6 control animals received no immunosuppression, 6 animals were treated with MMF from day 45 after transplantation on in an individual maximal tolerated dose (mean 99.2 mg/kg/day). Until day 45 the animals did not receive any immunosuppressive treatment. The progression of GVD was quantified by IVUS as changes in intimal volume (IV) in the midsegments of all grafts every 3 weeks until day 105 when the animals were euthanized and the grafts have been harvested for histopathological analysis. Pharmacokinetik and pharmacodynamic monitoring was used to optimize the immunosuppressive efficacy. ResultIn serial IVUS measurements IV progressed after day 42 from 25 ± 1 to 44 ± 4 on day 63 to 52 ± 4 on day 84 to 55 ± 3mm2 on day 105 in the control versus 25 ± 4, 40 ± 7, 43 ± 7 and 47 ± 7 mm2 in the treatment group (p = 0.3). Also the difference in IA failed to be significant overall between control and treatment group, there was a significant correlation between mean MMF dose and IA (r = − 0.88, P = 0.01): In four out of 6 animals no dose reduction due to toxicity was necessary and in all these animals IA was lower than in the control animals. In two animals with MMF toxicity and dose reduction, high IA was observed. ConclusionsIn this demanding model evaluating advanced graft vascular disease in non-human primates MMF was able to halt GVD when given in a high maximal tolerated dose. In case of toxicity and individual necessary dose reduction, progress of GVD was not altered.