Concentrations Of CsA Research Articles

ComparativeIn VitroEffects of Calcineurin Inhibitors on Functional Vascular Relaxations of Both Rat Thoracic and Abdominal Aorta

Background and Aim. Calcineurin inhibitors (CNIs) have shown to develop hypertension in transplant patients. The in vitro incubation effects of cyclosporine (CsA) and tacrolimus (Tac) on vascular relaxations of rat thoracic aorta (TA) and abdominal aorta (AA) need to be investigated. Methods. The optimal concentrations of CsA (1.0 mg/mL) and Tac (0.1 mg/mL) used to compare endothelium-dependent (acetylcholine (ACh)) and endothelium-independent (sodium nitroprusside (SNP)) vascular relaxation against the agonists in phenylephrine (PE-) constricted TA and AA of 13-week-old male Sprague Dawley rats (n = 6). Results. In TA, the maximal vasodilator response elicited by ACh (control: I max 98%) was significantly (P < 0.01) inhibited by CsA (I max 10%) but not by Tac (I max 97%). In AA, (control: IC50 50 nM; I max 100%) CsA (IC50 7 μM; (P < 0.01) showed strong sensitivity to inhibit ACh-dependent vascular relaxation than Tac (IC50 215 nM (P < 0.05); I max 98%). CsA and Tac failed to affect the inhibitory responses to SNP in both TA and AA. Conclusion. CsA exerts profound inhibitory effect on endothelium-dependent vasodilatation as compared to Tac in both TA and AA. Aortic rings from the thoracic region are more sensitive to CNIs, since the vasodilator response to ACh is solely mediated by NO while in the AA, ACh likely recruits other endothelial mediators besides NO to maintain vasodilatation.

Impact of CYP3A4*1B and CYP3A5*3 polymorphisms on the pharmacokinetics of cyclosporine and sirolimus in renal transplant recipients

Calcineurin inhibitor (cyclosporine, CsA) and mTOR inhibitors (sirolimus, SRL) - immunosuppressants used to prevent allograft rejection after renal transplantation - have a narrow therapeutic index and show considerable inter-individual pharmacokinetic differences. Differences in expression and activity of cytochrome P450 (CYP) 3A4 and 3A5 affect these pharmacokinetics; cytochrome activity differences are associated with CYP genetic polymorphisms. This study evaluated the effects of polymorphisms in CYP3A4 and CYP3A5 on immunosuppressive drug-dose adjusted trough blood concentrations. One hundred renal transplant recipients were genotyped for CYP3A4*1B and CYP3A5*3 using PCR-RFLP. Blood concentrations of CsA and SRL were determined by EMIT and HPLC/UV, respectively. The allelic frequencies of CYP3A4*1B and CYP3A5*3 in the study group were 2.5% and 96.5%, respectively. The mean cyclosporine dose in CYP3A4*1/*1B subjects was 455.04±128.68 mg/day vs. 261.68±64.72 mg/day in CYP3A4*1/*1 subjects (p<0.001). The mean cyclosporine dose-adjusted trough blood concentrations (ng/ml per mg/kg body weight) in CYP3A4*1/*1B subjects were lower than in the CYP3A4*1/*1 group (37.06±10.38 vs. 44.63±13.99; p<0.218). The mean cyclosporine dose in CYP3A5*1/*3 subjects was 400.65±164.97 mg/day vs. 263.52±64.39 mg/day in CYP3A5*3/*3 subjects (p<0.022). No association was detected between sirolimus trough blood concentration (C0) or dose requirement, and CYP3A4 or CYP3A5 genotype. Genetic polymorphisms in CYP3A4 and CYP3A5 may underlie inter-individual differences in cyclosporine pharmacokinetics after renal transplantation. Patients with at least 1 functional CYP3A5*1 or CYP3A4*1B allele require significantly higher doses of cyclosporine to reach target drug levels compared to patients with the CYP3A4*1 or CYP3A5*3 alleles.

Time‐course effects of St John’s wort on the pharmacokinetics of cyclosporine in dogs

To clarify the interaction between St John's wort (SJW) and cyclosporine (CsA) in dogs, the pharmacokinetics of CsA before and during the repeated administration of SJW were analyzed. In the SJW group, SJW (300 mg) was given orally to four dogs every 24 h for 14 days. A single dose of CsA (5 mg/kg) was given orally 7 days before and 7 and 14 days after the initiation of the repeated administration of SJW. In the Control group, a single dose of CsA (5 mg/kg) was given orally to four other dogs in accordance with that in the SJW group. Blood samples from both groups were collected, and whole-blood concentrations of CsA were determined using high-performance liquid chromatography with UV detection. The maximum whole-blood concentration and AUC(0-∞) of the SJW group were significantly lower and the CL(tot) /F and V(d) /F were significantly higher than those in the Control group 7 and 14 days after the initiation of repeated SJW. Thus, repeated administrations of SJW affect the pharmacokinetic profiles of CsA in dogs. Further studies are necessary to elucidate the mechanisms of interaction between SJW and CsA in dogs.

Age and sex differences in the effects of the immunosuppressants cyclosporine, sirolimus and everolimus on rat brain metabolism

Application of the widely used immunosuppressant (ISS) cyclosporine (CsA) is severely limited by a number of serious side-effects such as kidney and neurotoxicity. As we have shown before, CsA exhibits metabolic toxicity in brain-models. The macrolide ISSs sirolimus (SRL) and everolimus (RAD) are capable of modulating these CsA-induced effects. It was our aim to study the age-dependent metabolic changes in the rat brain after ISS-treatment and the possible role of the blood–brain-barrier in modulation of CsA metabolic toxicity.Young and adult rats were treated orally with one ISS alone or in combination with CsA for six days. Metabolic changes were assessed by nuclear magnetic resonance (NMR) spectroscopy of brain extracts as toxicodynamic endpoints. Brain P-glycoprotein (P-gp) and ISS concentrations were determined as pharmacokinetic endpoints. Young rats were more susceptible to CsA-induced inhibition of the Krebs cycle (glutamate: 78% of controls, glutamine: 82%, GABA: 71% in young vs. 85%, 89%, 92% in adult rats). Increased glycolysis after CsA-treatment was sufficient to maintain the energy state at control levels in adult brains, but not in the young rat brains (phosphocreatine: 35%). Tissue concentrations of CsA and SRL within the brain of young rats were three-fold higher, while concentrations of P-gp were three-fold higher in adult rat brains. Our results suggest that age-dependent differences in the blood–brain barrier led to increased ISS brain concentrations and hence inhibition of brain energy metabolism.

Impact of Cyclosporine-A Concentration on the Incidence of Severe Acute Graft-versus-Host Disease after Allogeneic Stem Cell Transplantation

This single-center retrospective study analyzed 85 consecutive patients who underwent allogeneic stem cell transplantation (allo-SCT) with the aim to assess whether there is a correlation between exposure to cyclosporine-A (CsA; as measured by CsA concentrations during the first month after allo-SCT) and the risk for developing severe grade III-IV acute graft-versus-host disease (aGVHD). The median concentrations of CsA in the blood at 1, 2, 3, and 4 weeks after allo-SCT were 348 (range: 172-733), 284 (range: 137-535), 274 (range: 107-649), and 247 (range: 37-695) ng/mL, respectively. Overall, grade II-IV aGVHD occurred in 36 patients (42%) at a median of 29 (range: 6-100) days after allo-SCT. The incidence of grade III-IV aGVHD (n = 20) was 23% (95% confidence interval [CI], 14%-32%). In univariate analysis, patients receiving allo-SCT from an HLA-matched unrelated donor had a higher risk of grade III-IV aGVHD, and patients having the lowest CsA concentration in the first and second weeks after allo-SCT had a significantly higher risk of grade III-IV aGVHD. In a multivariate logistic regression analysis, a higher CsA concentration measured during the first week following graft infusion was the strongest parameter significantly associated with a reduced risk of severe grade II-IV aGVHD (P = .012; relative risk [RR] = 0.24; 95% CI, 0.08-0.73). Of note, when adjusted by donor type, CsA concentration in week 1 remained significantly associated with risk of severe grade II-IV aGVHD (P = .014). We conclude that precise monitoring of CsA concentrations and adjustment of CsA dose early after allo-SCT may be effective to prevent onset of severe aGVHD.

Clinical Application of Cordyceps sinensis on Immunosuppressive Therapy in Renal Transplantation

Objective We sought to explore the adjunctive effects of Cordyceps sinensis (CS) in clinical renal transplantation. Materials and Methods Patients (n = 202) were divided randomly by lottery into a treatment (n = 93) and a control group (n = 109). Patients in the treatment group were treated with CS 1.0 g 3 times a day in addition to the immunosuppressive regimen given to the control group. We compared patient and graft survivals, incidence, time and severity of acute rejection episodes, chronic allograft nephropathy (CAN), hepatotoxicity and nephrotoxicity, biochemistry parameters including indicators of liver and kidney functions, fats, proteinuria, dosages, and whole blood concentrations of cyclosporine (CsA). Results Patient and graft survival rates, serum creatinine (SCr), and blood urea nitrogen (BUN) were not significantly different between the 2 groups ( P > .05). Serum uric acid (UA) and 24-hour urinary total protein (24-hour UTP) were significantly lower in the treatment group than in the control group ( P < .05). The incidences (11.83% vs 15.60%) and times to acute renal allograft rejection (23.48 ± 7.22 vs 22.27 ± 8.03 days posttransplantation) were not significantly different between the treated and control groups ( P > .05). Patients receiving thymoglobulin antirejection therapy (3 cases) were fewer in the heated versus control group (13 cases; P = .014). The incidences of hepatotoxicity and nephrotoxicity in the treated group were 12.90% and 19.35%, significantly lower than 24.77% and 33.94% in the control group, respectively ( P < .05). At 2 to 6 months posttransplantation, the CsA dosages in the treated group were significantly lower than those in the control group ( P < .05). The whole blood trough CsA concentrations in the treated group were significantly lower than those in the control group at 3 to 6 months posttransplantation ( P < .05). The decreasing trends of the 2 aforementioned parameters in the treatment group were approximately linear among treated subjects compared with approximately quadratic in the control group ( P < .05). The incidence of CAN in the treated group was 7.53%, which was significantly lower than 18.35% in the control group ( P = .024). The 24-hour UTP level in CAN patients within the treated group was significantly lower than the control group after transplantation ( P = .045). The differences in total bilirubin, SCr, serum UA, and total cholesterol levels among otherwise normal patients in the treated group were significantly lower than those among the control group ( P < .05). Conclusions The use of CS may allow decreased dosages and concentrations of CsA causing fewer side effects without an increased risk of acute rejection. In addition, CS with reduced dose CsA may decrease proteinuria and retard CAN progression.

Genetic Polymorphisms in CYP3A5 and MDR1 Genes and Their Correlations With Plasma Levels of Tacrolimus and Cyclosporine in Renal Transplant Recipients

Immunosuppressive drugs, such as tacrolimus (FK506) and cyclosporine (CsA), play an essential role in graft survival, preventing rejection. Large interindividual differences in drug-metabolizing enzymes as well as in drug transporters make the task of reaching the optimal concentrations difficult. The bioavailability of CsA and FK506 seems to be associated with the cytocrhome P450 IIIA ( CYP3A) gene. It has also been described that the Multi Drug Resistance 1 ( MDR1) gene that encodes for polyglycoprotein-P (P-gp) may influence the metabolizing action of FK506 and CsA. Therefore, we sought, to correlate single nucleotide polymorphisms (SNPs) in the CYP3A and MDR1 genes with the concentrations of FK506 and CsA. For this purpose we analyzed 2 groups of renal transplant recipients by sequencing: one receiving a CsA immunosuppressive regime, and other, an FK506-immunosuppression. This study showed that subjects in the FK506 group who had encoded the 1236C>T substitution in the MDR1 gene displayed 44.4% higher drug concentrations compared with (“wild-type”) individuals. Individuals carrying the 2677G>T,A mutation showed FK506 concentrations that were 44.7% higher than the wild-type individuals. Concerning the CsA group, individuals carrying the 22915A>C substitution displayed CsA concentrations 52.1% higher than wild-type individuals.

Intrarenal activation of renin angiotensin system in the development of cyclosporine A induced chronic nephrotoxicity

The relationship between cyclosporine-induced chronic nephrotoxicity (CAN) and renin-angiotensin II in humans is still contradictory. This study was conducted to detect the levels of renin and angiotensin II (ANGII) both in renal tissue and plasma from kidney transplantation patients suffering from CAN. Twenty-six patients with allograft biopsy-proven CsA-related chronic nephrotoxicity (CAN group) and chronic rejection (control group) were enrolled in this study. Renal tissues were subjected to immunohistochemical staining with renin and ANGII antibodies. Renin and ANGII plasma levels were measured when the biopsy was performed. The relationship between expression of renin or ANGII and clinicopathological manifestations were also investigated. The cyclosporine plasma level was obtained 2 hours after morning dose (C2). In vitro, human umbilical vein endothelial cells (HUVEC) and rat mesangial cells (MC) were incubated with different concentrations of CsA (0, 250, 500, 1000 microg/L) for 24 hours. Secretion and expression of renin and ANGII was measured by radioimmunoassay or immunohistochemical staining. Renal pathological scores for renin and ANGII expression were significantly higher in specimens of CAN than in controls (P < 0.05). The plasma levels of renin, ANGII and C(2) in the CAN group were higher than the control group, but no significant difference was found ((0.37 +/- 0.12) ng x ml(-1)x h(-1) vs (0.20 +/- 0.10) ng x ml(-1) x h(-1), P = 0.076; (122.69 +/- 26.73) pg/ml vs (121.88 +/- 36.35) pg/ml, P = 0.977; (719.04 +/- 55.89) ng/ml vs (658.80 +/- 90.78) ng/ml, P = 0.196, respectively). In vitro, renin as well as ANGII expression increased significantly in both HUVEC and MC after the cells were incubated with CsA for 24 hours (P < 0.05). CsA also stimulated the secretion of ANGII in HUVEC and MC in a dose-dependent manner. Renal allograft biopsy is important to differentiate chronic CsA-related nephropathy from chronic rejection. The intrarenal renin angiotensin system plays an important role in CsA-related chronic nephropathy. The histological lesions of CsA nephrotoxicity fail to correspond spontaneously to either the change of C2 level or the change of renin and ANGII plasma level. CsA stimulates the secretion of ANGII and the expression of renin and ANGII in HUVEC and MC. Blockage of RAS may be helpful for therapeutic intervention in the progression of CsA-related chronic nephropathy.

An In Vitro System for the Determination of Individualized Immunosuppression

The risk of complications of immunosuppressive treatment in organ transplantation increases with the aggregate amount of immunosuppressive medication given to the patient. As the doses of immunosuppressive agents required to achieve comparable effects show considerable variability, methods to assess individual sensitivity toward immunosuppressive regimens are urgently needed. The aim of this study was to develop such an in vitro test system. As immunological model for allogeneic transplantation, individual pairs of recipient-derived lymphocytes and of donor-derived B lymphocytes mimicking HLA expression of cells in the transplanted organ were isolated and assessed in mixed-lymphocyte cultures (MLC). Alloreactivity was readily observed and MLC consisted of CD8 + and CD4 + T cells as well as CD56 + natural killer cells. A proliferation assay to measure the response of individual MLC on the immunosuppression by cyclosporine (CsA) was developed. The concentrations of CsA leading to growth reductions by 50% (inhibitory concentration 50, IC 50) were found between 110 and 220 ng/mL, which was near the trough whole blood levels for CsA. Accordingly, the IC 90 values (660 to 1760 ng/mL) were near the target values for peak whole blood levels. We believe that these data present a simple and potentially useful in vitro technology that allows for the prediction of individual responses to immunosuppressive therapeutic regimens.

Do MMP-1 Levels of Gingival Fibroblasts Have a Role in the Gingival Overgrowth of Cyclosporine-Treated Patients?

The aim of this study was to compare the matrix metalloproteinase-1 (MMP-1) levels in gingival fibroblast cultures derived from two groups of renal transplant patients receiving cyclosporine (CsA) who exhibit gingival overgrowth and who have healthy periodontium. Gingival fibroblasts obtained from four patients with CsA-induced gingival overgrowth (CsA-GO) and four patients who receive CsA but have healthy periodontium were incubated with increasing concentrations of CsA and cultured for 72 hours. Expression levels of MMP-1 in all the groups were measured four times at 0, 24, 48, and 72 hours by the Rapid Collagenase Assay Kit. No significant difference was seen at baseline. As the CsA concentration and the duration in the cell media increased, the CsA-GO showed that fibroblasts displayed significantly suppressed MMP-1 levels with respect to the baseline, at which fibroblasts from CsA patients with healthy periodontium exhibited the same result as at the highest CsA concentration. Results of this study indicated that CsA therapy did not have a significant effect on MMP-1 levels. Since the overall pathogenesis of drug-induced gingival hyperplasia has been accepted as multifactorial, down-regulation of MMP-1 expression may play a minor role.

Protein Phosphatase 2B Inhibitor, Cyclosporine A, Stimulates Endothelial Cell Exocytosis of Ultra-Large VWF Multimeric Strings by Regulating Munc18c Phosphorylation.

Exocytosis of Weibel Palade bodies (WPBs) containing ultra-large Von Willebrand factor (ULVWF) plays an essential role in the processes of inflammation and thrombosis. The exocytosis of ULVWF involves the fusion of WPBs to the endothelial cell plasma membrane via vesicular trafficking families of proteins. Reversible post-translational modification, including the phosphorylation of tyrosine (Tyr) and/or serine/threonine (Ser/Thr) residues or S-nitrosylation of cysteine residues on one or more vesicular trafficking proteins tightly regulates the exocytotic process. The phosphorylation of any trafficking protein is determined by the interplay between protein kinases and protein phosphatases and any alteration in the activity of either enzyme can affect exocytosis. In comparison to agonist-induced, protein kinase-dependent signaling pathways that regulate exocytosis in endothelial cells, there is scant information about any possible role for the regulation of protein phosphatases in this process. Inhibition of protein phosphatase 2B (PP2B) also called calcineurin by the immunosuppressive drug, cyclosporine (CsA), is associated with thrombotic microangiopathy in a subset of transplant patients. Calcineurin is the target of a cyclophilin-CsA complex. We detected both calcineurin and cyclophilin A in resting human umbilical vein endothelial cells (HUVECs). Because CsA is a potent inhibitor of PP2B, we hypothesized that CsA induces ULVWF secretion and thrombotic events at least in part, by facilitating the phosphorylation of vesicle trafficking proteins that promote the exocytosis of ULVWF from endothelial cells. In vitro, we found that nanomolar concentrations of CsA or a cell permeable PP2B auto inhibitory peptide stimulated the release of ULVWF from HUVECs. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) viability assays revealed that PP2B inhibitors did not damage the HUVECs. In vivo, plasma levels of VWF were increased after intraperitoneal injection of CsA. These studies suggest that PP2B negatively regulate ULVWF exocytosis. Microscopic studies revealed that the ULVWF strings secreted by HUVECs treated with CsA or PP2B auto-inhibitory peptide induced the adhesion of platelets. CsA-treated HUVECs exhibited an increased serine phosphorylation of Munc18c, a vesicle trafficking associated protein that promotes granule exocytosis. These findings support a mechanism whereby inhibition of PP2B by CsA enhances cell secretion, including endothelial cell secretion of ULVWF by facilitating Munc18c phosphorylation.

Apoptosis by Cyclosporine in Mesangial Cells

Introduction The immunosuppressive agent cyclosporine (CsA) is widely used to treat allograft rejection and various autoimmune disorders. A major limiting factor in the use of CsA is chronic nephrotoxicity. The pathogenesis of CsA-induced nephrotoxicity is not fully understood. Several recent studies have suggested that CsA treatment directly induces apoptosis in several cell types. The present study was undertaken to investigate the effects of CsA on apoptosis of cultured rat mesangial cells (RMCs). Methods RMCs were treated with CsA at concentrations of 0.1 to 40 μmol/L. Cell viability was determined by MTT assay. Apoptotic protein expression was determined by Western blot analysis. Results Cell viability was decreased with increasing concentrations of CsA in dose-dependent manner. CsA produced dose-dependent induction of p53, caspase-6, and Bax protein expression. CsA treatment caused proteolytic cleavage of caspase-3 and induced the degradation of 116-kDa PARP into 89-kDa fragment. RMCs with CsA reduced Bcl-2 and cIAP expression. Conclusions In this study, CsA induced apoptosis by up-regulating proapoptotic factors, caspase-3 and -6, p53, Bax, cleaving PARP, and down-regulating antiapoptotic factor, Bcl-2, and cIAP. These results suggested that the increased cell apoptosis exerted by CsA may be one of the mechanisms promoting CsA-induced nephrotoxicity.

Effects of Cyclosporine and Tacrolimus on the Oxidative Stress in Cultured Mesangial Cells

Objectives Cyclosporine (CsA) and tacrolimus (Tac) are two primary immunosuppressive agents used for the prevention of graft rejection. However, their use is associated with significant side effects, most notably nephrotoxicity. The mechanisms of this toxicity are not fully understood, but they seem to be associated with increases in the production of oxygen free radicals (OFRs). This present work examined the effect of CsA and Tac on the production of OFRs in cultured rat renal mesangial cells (RMCs). Methods Varying concentrations of CsA and Tac (0 to 40 μmol/L) were added to RMCs and incubated for 60 minutes at 37°C. The production of OFRs was evaluated by measuring the fluorescent product from the oxidation of an oxidant-sensitive 2′, 7′-dichlorofluorescin. Results At 60 minutes, the relative fluorescence units (RFU) for OFRs production in RMCs exposure to CsA were increased by 2.5%, 11.5%, 22.5%, 57.2%, and 174% at 2.5, 5, 10, 20, and 40 μmol/L, respectively. Tac increased the RFU by 15.9%, 13.6%, 14.8%, 13.2%, 21.4%, 13.2%, and 28.1% at 0.1, 1, 2.5, 5, 10, 20, and 40 μmol/L, respectively. In RMCs, the RFU produced by CsA was higher than that by Tac. Conclusions The results of this experiment suggest that CsA and Tac induced renal injury by OFRs.

Camel (Camelus dromedarius) immunoglobulin G, α-lactalbumin, serum albumin and lactoferrin in colostrum and milk during the early post partum period

Colostrum and milk samples from twelve Tunisian camels were analysed for concentration of immunoglobulin G (IgG), alpha-lactalbumin (alpha-la), serum albumin (CSA) and lactoferrin throughout the first 14 milkings post partum (7 days of lactation) using single radial immunodiffusion assay. Concentrations (mg/ml, means+/-SD) at first milking were IgG, 100.7+/-60.4; alpha-la, 2.2+/-0.7; CSA, 8.5+/-3.6 and lactoferrin, 1.2+/-0.3. Large variations were recorded for IgG and CSA concentrations (11.8-211.1 mg/ml and 2.9-13.8 mg/ml respectively) Concentrations of IgG and CSA dropped abruptly in the subsequent milkings while alpha-la concentration increased until milking 5 and then decreased slowly. Lactoferrin dropped only from milking 7. Mean IgG concentrations were 3.6 and 2.5 mg/ml at milking 9 and 13 respectively. However, IgG concentration did not differ significantly, at the 1% level, from milkings 11 to 14. The contribution of CSA to the increase in whey proteins in early milks was greater than that described in the bovine and caprine species.

Regulation of Ca2+-dependent Desensitization in the Vanilloid Receptor TRPV1 by Calcineurin and cAMP-dependent Protein Kinase

The vanilloid receptor TRPV1 is a polymodal nonselective cation channel of nociceptive sensory neurons involved in the perception of inflammatory pain. TRPV1 exhibits desensitization in a Ca2+-dependent manner upon repeated activation by capsaicin or protons. The cAMP-dependent protein kinase (PKA) decreases desensitization of TRPV1 by directly phosphorylating the channel presumably at sites Ser116 and Thr370. In the present study we investigated the influence of protein phosphatase 2B (calcineurin) on Ca2+-dependent desensitization of capsaicin- and proton-activated currents. By using site-directed mutagenesis, we generated point mutations at PKA and protein kinase C consensus sites and studied wild type (WT) and mutant channels transiently expressed in HEK293t or HeLa cells under whole cell voltage clamp. We found that intracellular application of the cyclosporin A.cyclophilin A complex (CsA.CyP), a specific inhibitor of calcineurin, significantly decreased desensitization of capsaicin- or proton-activated TRPV1-WT currents. This effect was similar to that obtained by extracellular application of forskolin (FSK), an indirect activator of PKA. Simultaneous applications of CsA.CyP and FSK in varying concentrations suggested that these substances acted independently from each other. In mutation T370A, application of CsA.CyP did not reduce desensitization of capsaicin-activated currents as compared with WT and to mutant channels S116A and T144A. In a double mutation at candidate protein kinase C phosphorylation sites, application of CsA.CyP or FSK decreased desensitization of capsaicin-activated currents similar to WT channels. We conclude that Ca2+-dependent desensitization of TRPV1 might be in part regulated through channel dephosphorylation by calcineurin and channel phosphorylation by PKA possibly involving Thr370 as a key amino acid residue.

Are Anemic Patients More Sensitive to Calcineurin Inhibitors?

A calcineurin inhibitor (CNI) is characterized by high affinity binding to red blood cells. There is a possibility that hematocrit levels might affect the immunosuppressive effects of cyclosporine (CsA). The purpose of this study was to examine whether the treatment with CNI was more effective in preventing acute rejection in anemic compared with nonanemic patients and to find a suitable method to monitoring immunosuppression. Ninety-five living donor renal transplant recipients who were treated with CsA were divided into five groups depending on their Ht levels. The incidences of biopsy-proven acute rejection were 1/8 (12.5%), 8/31 (25.8%), 6/28 (21.4%), 10/22 (45.5%), and 2/6 (33.3%) for Ht ≤ 25%, 25% < Ht ≤ 30%, 30% < Ht ≤ 35%, 35% < Ht ≤ 40%, and 40% < Ht, respectively. In vitro IL-2 mRNA inhibition tests were performed to evaluate lymphocyte function after stimulation of whole-blood with phorbol myristate acetate and Ca-ionophore in the presence of various concentrations of CsA. Whole blood CsA levels causing 50% inhibition of IL-2 mRNA (IC50) were 256, 310, 175, and 55 ng/mL for Ht 50%, 40%, 30%, and 20%, respectively. It is speculated that plasma concentrations of CsA may increase at low Ht levels, because lower incidences of acute rejection and lower IC50 values of CsA were observed in the anemic state. When the dosage of CsA was adjusted to its whole-blood concentration, the anemic state is likely to enhance the immunosuppressive effect of CsA. A pharmacodynamic study, such as the IL-2 mRNA inhibition test, is preferable for CsA monitoring.

Concentrations of cyclosporin A and FK506 that inhibit IL-2 induction in human T cells do not affect TGF-β1 biosynthesis, whereas higher doses of cyclosporin A trigger apoptosis and release of preformed TGF-β1

Cyclosporin A (CsA) and FK506 suppress T cell activation by inhibiting calcineurin and the calcineurin-dependent transcription factors nuclear factor of activated T cells (NFATc), which are central regulators of T cell function. It was reported that CsA up-regulated the transcription of transforming growth factor-beta1 (TGF-beta1) in lymphocytes and other cells and activated its promoter in A549 lung carcinoma cells, but the mechanisms involved are poorly understood, and it is unclear whether calcineurin plays any role. We have studied the regulation of TGF-beta1 in normal human lymphocytes and cell lines. In Jurkat T cells, the TGF-beta1 promoter was activated by calcineurin and NFATc and inhibited by CsA and FK506. However, the promoter was insensitive to both drugs in A549 cells. In human T cells preactivated with phytohemagglutinin, biosynthesis of TGF-beta1, induced by the T cell receptor (TCR) or the TGF-beta receptor, was not substantially affected by CsA and FK506 concentrations (< or = 1 microM) that effectively inhibited interleukin-2 production. However, pretreatment of fresh lymphocytes with CsA or FK506 during primary TCR stimulation reduced their production of TGF-beta1 during secondary TCR activation. Finally, high concentrations of CsA (10 microM), in the range attained in vivo in experiments in rodents, caused apoptosis in human T cells and the release of preformed, bioactive TGF-beta1. These effects are unlikely to owe to calcineurin inhibition, as they were not observed with FK506. Our results indicate that CsA and FK506 are not general inducers of TGF-beta1 biosynthesis but can cause different effects on TGF-beta1 depending on the cell type and concentrations used.

LOW INCIDENCE OF MALIGNANCY AMONG RENAL TRANSPLANT RECIPIENTS TREATED AT A SINGLE CENTER FOR 1–10 YEARS WITH A SIROLIMUS-CYCLOSPORINE REGIMEN

O90* Aim: Previous multicenter studies of neoplasms among renal transplant recipients treated with a sirolimus (SRL)-cyclosporine (CsA)-prednisone regimen were limited to 24-mo follow-up. In contrast, we have examined the courses of 1008 patients treated at a single center for up to 10 yr. Methods: The single-center experience includes patients in the Phases I–IV development of sirolimus, using doses ranging from 1–7mg/m2 and CsA at C2 exposures of 200–1200ng/mL with limited steroid courses in about 35% of patients. The follow-up (mean±SD) of 60.3±27.5 mo was exclusively at the medical center throughout their course. Routine queries and examinations specifically probed the occurrence of skin neoplasms. The Paradox database was compared with literature sources and with the percentages in the US population as described in the Surveillance, Epidemiology, and End Results (SEER) database using chi-square analysis for the incidences of various neoplasms. Results: The overall incidence of malignancy throughout follow-up was 34/1008 (3%) with presentation at 34.8±30.1 mo (range: 1–135). The demographics of the 33 affected patients (1 with 2 malignancies) were men:women=28:5 with a mean age at presentation of 53.0±12.5 years. The incidence of various types of malignancies common to transplant patients was lymphoproliferative disorder (PTLD) 0.4%, renal cell carcinoma (RCC) 0.2%, and skin tumors 1.9%, including squamous cell (0.9%), basal cell (0.5%), melanoma (0.2%), Merkel cell (0.2%), and basosquamous (0.1%). The distribution of tumor types among malignancy-positive patients was similar to transplant registry figures for skin tumors (63.3% vs 43.1%), PTLD (13.3 vs 12.2%), and RCC (6.7 vs 4.3%). The other malignancies included single cases of breast, bladder, endometrial, and brain neoplasms, as well as two cases of lung neoplasms. Furthermore, when our data were compared to SEER over a 5-yr period, the SRL-CsA cohort showed a similar incidence of skin tumors (1.9 vs 1.5%), which is significantly less than the 7% reported among other renal transplant patient cohorts. Compared to the general US population, our patients showed a 4-fold increase in PTLD (0.4 vs 0.1%) and RCC (0.2 vs 0.05%), figures that were far less than the previously reported 27.2-and 8-fold increases, respectively, using tacrolimus plus mycophenolate. At the time of diagnosis, the trough concentrations (mean±SD) of SRL (11.4±8.9ng/mL) and CsA (138.75±84.1) in affected patients were similar to those of other recipients and not excessive. The treatment outcomes were satisfactory save for 5 deaths: PTLD in a renal allograft incidentally discovered at the time of demise, endometrial carcinoma in a 52-year-old woman, lung cancer in a 72-year-old smoker, brain tumor in a 73-year-old man, and leukemia in a 24-year-old man. Four other graft losses included 2 lethal cardiovascular events and 2 chronic rejections due to reduced immunosuppression. Conclusion: In renal allograft recipients, SRL seems to reduce the incidence of tumors, particularly of the skin, suggesting an antineoplastic effect.

The effects of the calcineurin inhibitors cyclosporin and ascomycin on an in-vitro assay for autoimmune urticaria

Rationale Since the calcineurin inhibitor, cyclosporin, has been used successfully in the treatment of chronic nonallergic urticaria, we examined the effect of cyclosporin (CSA) and ascomycin (ASM) on the release of histamine from basophils when stimulated with sera from patients with chronic autoimmune urticaria. Methods Healthy volunteer leukocytes were isolated and incubated in varying concentrations of CSA and ASM (0.1–10000nM) for 30 minutes prior to stimulation with serum from patients with urticaria known to have functional IgG antibodies directed against the alpha subunit of the IgE receptor. Results Pre-incubating cells with CSA and ASM produced dose-dependent inhibition of histamine release from sera of urticaria patients. Inhibition was not prevented by C5a receptor blocking antibodies. Dose-dependent inhibition was seen to purified IgG from serum but not from complement C5a. Conclusions Calcineurin inhibitors such as CSA and ASM may work by interfering with intracellular signalling in basophils and mast cells following cross-linking of the IgE receptor but not by stimulation of the C5a receptor.

Assessment of immunosuppressive drug interactions: inhibition of lymphocyte function in peripheral human blood

Cyclosporin (CsA) or tacrolimus (TRL) is routinely combined with either sirolimus (SRL) or mycophenolate mofetil (MMF) in immunosuppressive regimes in organ transplantation. The aim of our study was to establish a specific human blood assay of lymphocyte function in order to assess interactions of these drug combinations. Different concentrations (10 6–10 9 nM) of CsA, TRL, SRL or mycophenolic acid (MPA, the active metabolite of MMF) was added to whole blood of five human volunteers. Drug combinations were studied by adding 250, 500 or 1000 nM of MPA to different concentrations of CsA, TRL, or SRL or by adding 1, 10 or 25 nM of SRL to different concentrations of CsA or TRL. After concanavalin-A stimulation, whole blood cultures were analyzed by flow cytometry detecting lymphocyte proliferation and activation by bivariate expression of proliferating cell nuclear antigen (PCNA)/DNA content and T cell-surface activation antigens (e.g. CD25, CD95, and CD154). We found an order of potency inhibiting lymphocyte function with SRL>TRL>CsA>MPA. In addition, we observed enhanced inhibition of PCNA, CD25, CD95 or CD154, if either CsA or TRL was combined with low concentrations of MPA, or SRL alone or if SRL was combined with low concentrations of MPA. Data analysis revealed an independent functional synergism or partial agonism in most combinations. This human blood assay is able to assess lymphocyte function and to monitor immunosuppressive therapy. The assay also permits pharmacological analysis of drug interactions, which will lead to improved safety and therapeutic efficacy in transplanted patients.