High-dose Cyclosporin A Research Articles

Efficacy and Safety of Low-Dose Cyclosporine Relative to Immunomodulatory Drugs Used in Atopic Dermatitis: A Systematic Review and Meta-Analysis

Cyclosporine A (CsA) is effective in treating moderate-to-severe atopic dermatitis (AD). This systematic review and meta-analysis aimed to summarize the effectiveness and safety of low-dose (<4 mg/kg) versus high-dose (≥4 mg/kg) CsA and other systemic immunomodulatory agents in patients with AD. Five randomized controlled trials met the inclusion criteria. The meta-analysis included 159 patients with moderate-to-severe AD who were randomized to receive low-dose CsA, and 165 patients randomized to receive high-dose CsA and other systemic immunomodulatory agents. We found that low-dose CsA was not inferior to high-dose CsA and other systemic immunomodulatory agents in reducing AD symptoms [standard mean difference (SMD) -1.62, 95% confidence interval (CI) -6.47; 3.23]. High-dose CsA and other systemic immunomodulatory agents showed a significantly lower incidence of adverse events [incidence rate ratio (IRR) 0.72, 95% CI 0.56; 0.93], however, after sensitivity analysis, there was no difference between the two groups except for one study (IRR 0.76, 95% CI 0.54; 1.07). Regarding serious adverse events requiring discontinuation of treatment, we observed no significant differences between low-dose CsA and other systemic immunomodulatory agents (IRR 1.83, 95% CI 0.62; 5.41). Our study may justify the use of low-dose CsA rather than high-dose CsA and other systemic immunomodulatory agents in moderate-to-severe AD.

Efficacy and safety of low-dose cyclosporine relative to immunomodulatory drugs used in atopic dermatitis: A systematic review and meta-analysis

Cyclosporine A (CsA) is effective in treating moderate-to-severe atopic dermatitis (AD). However, several side effects have been reported. This systematic review and meta-analysis aimed to summarize the effectiveness and safety of low-dose versus high-dose CsA and other systemic immunomodulatory agents in patients with AD.

Intravenous immunoglobulins, cyclosporine, and best supportive care in epidermal necrolysis: Diverse effects on systemic inflammation.

Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are rare but potentially life-threatening cutaneous adverse reactions. There is still no consensus on adjuvant treatments, and little is known about their effects on systemic inflammation in SJS/TEN. Our aim was to characterize the systemic and cutaneous immune profiles of SJS/TEN patients and to investigate whether/how intravenous immunoglobulins (IVIG), cyclosporine A (CSA), and best supportive care only (BSCO) affected the systemic immune signature and clinical outcome (6week-mortality, complications, hospitalization stay). We included 16 patients with SJS/TEN, treated with high-dose IVIG (n=8), CSA (n=4) or BSCO (n=4). Serial serum samples were obtained prior-, 5-7 days, and 21 days after treatment onset. Serum levels of inflammation-/immune response-associated proteins were measured by high-throughput proteomics assay (OLINK) and cytotoxic molecules by ELISA. RNA extracted from skin biopsies collected prior treatment was analyzed by Nanostring. Serum inflammatory profiles in SJS/TEN patients were notably characterized by massive upregulation of type 1 immune response and proinflammatory markers. Surprisingly, there was limited overlap between cutaneous and serum immune profiles. Serial serological measurements of immune response markers showed very diverse dynamics between the different treatment groups. IVIG-treated patients showed completely different dynamics and most significant proteomic changes in an early phase (Day 5-7). In all treatment groups, type 1-/inflammatory response markers were dampened at day 21. Clinically, there were no outcome differences. Our study demonstrates that BSCO, CSA, and IVIG have very diverse biological effects on the systemic inflammatory response in SJS/TEN, which may not correlate with clinical outcome differences.

The effect of cyclosporin a on ischemia-reperfusion damage in a mouse model of ischemic stroke

ABSTRACT Objectives We aimed to investigate the protective effects of cyclosporin A (CsA) against ischemia-reperfusion (I/R) damage in a mouse ischemia model and the possible underlying mechanism. Methods Mice were divided equally into five groups: Sham, I/R, Vehicle, I/R plus CsA (10 mg/kg), and I/R plus CsA (20 mg/kg). Nerve function scores, infarct volume, brain water content, and Evans blue (EB) leakage were evaluated, and western blotting was performed to analyze the changes in CypA, p-Akt, NF-κB, MMP-9, and Claudin-5 expression. Results CsA can attenuate I/R damage in a mouse ischemic stroke model, as indicated by improved neurological function scores and decreased infarct volume, brain water content, and EB leakage. Additionally, high-dose CsA showed better protective effects than low-dose. The molecular mechanisms underlying the effects of CsA were explored, and it was found that CsA could inhibit the increase in CypA, p-Akt, NF-κB, and MMP-9 protein expression after middle cerebral artery occlusion, while Claudin-5 expression was decreased. Discussion CsA showed potential as a neuroprotective drug for the treatment of ischemic stroke patients; besides interfering with the typical NF-κB signaling pathway, the Akt pathway may also be involved in the effects of CsA.

A Randomized, Controlled Trial of Cyclosporine A Cationic Emulsion in Pediatric Vernal Keratoconjunctivitis: The VEKTIS Study

Vernal keratoconjunctivitis (VKC) is a chronic, allergic, and potentially severe ocular disease affecting children and adolescents that can lead to impaired quality of life (QoL) and loss of vision. This study evaluated the efficacy and safety of an investigational therapy for severe VKC, cyclosporine A (CsA) cationic emulsion (CE), an oil-in-water emulsion with increased bioavailability versus conventional CsA formulations. The VErnal KeratoconjunctiviTIs Study (VEKTIS) is a phase 3, multicenter, double-masked, vehicle-controlled trial. Pediatric patients (4 to younger than 18 years) with active severe VKC (grade of 3 or 4 on the Bonini severity scale) and severe keratitis (corneal fluorescein staining [CFS] score of 4 or 5 on the modified Oxford scale). One hundred sixty-nine patients were randomized to CsA CE 0.1% (1 mg/ml) eye drops 4 times daily (high dose), CsA CE twice daily (low dose) plus vehicle twice daily, or vehicle 4 times daily for 4 months. The primary end point was a mean composite score that reflected CFS, rescue medication use (dexamethasone 0.1% 4 times daily), and corneal ulceration over the 4 months. Differences in least-squares means versus vehicle for the primary end point were statistically significant for both the high-dose (0.76; P= 0.007) and the low-dose (0.67; P= 0.010) groups, with treatment effect mainly driven by CFS score. Significant differences were found between both active treatment groups and vehicle for use of rescue medication. Vernal keratoconjunctivitis symptoms and patient QoL (assessed by visual analog scale and the Quality of Life in Children with Vernal Keratoconjunctivitis questionnaire) improved in all 3 groups, with significant improvements for high-dose CsA CE versus vehicle. The efficacy of high-dose CsA CE in improving keratitis, symptoms, and QoL for those with severe VKC was demonstrated in these study patients. In addition, in this study cohort, CsA CE was well tolerated.

Effects Following Intracerebroventricular Injection of Immunosuppressant Cyclosporine A On Inhibitory Avoidance Learning and Memory in Mice

Background: Protein phosphatase-2B or calcineurin (CN) is the main phosphatase and a critical regulator of cellular pathways for learning, memory, and plasticity. Cyclosporine A(CyA), a phosphatase and peptidyl-prolyl cis/trans isomerase inhibitor, is a common immune suppressant extensively used in tissue transplantation. To further clarify the role of CN in different stages of learning and memory, the aim of the present study was to evaluate the role of CyA in an inhibitory avoidance (IA) model in mice.Materials and Methods: Using intracerebroventricular (ICV) injection of different doses of CyA (0.5, 5, and 50 nM) at different periods (pre-/post-training and pre-test), the effect of the drug was evaluated in a step-down IA paradigm. The latency of step-down (sec) was considered a criterion for memory performance. Results: The pre-training injections of CyA (0.5, 5 nM), however not of 50 nM, impaired IA learning acquisition. The post-training injection of high-dose CyA (50 nM) impaired memory consolidation. The pre-test ICV CyA injection did not impair memory retrieval; the ICV injection of CyA caused no change in locomotion. Conclusion: These findings suggest that CyA selectively interferes with acquisition, retention, but not retrieval, of information processing in mice. Given the crucial role of CN in common signaling pathway of memory performance and cognition, it could be a probable therapeutic target in the treatment of a wide variety of neurological conditions involving memory. [GMJ.2018;7:e1044]

High-Dose Subconjunctival Cyclosporine A Implants Do Not Affect Corneal Neovascularization after High-Risk Keratoplasty

To test whether subconjunctival cyclosporine A (CsA) implants affect the incidence and the degree of corneal neovascularization occurring after penetrating keratoplasty. Prospective, randomized, multicenter, controlled phase 2/3 clinical trial. The study comprised 43 trial sites in Germany, India, and the United States. Enrolled patients (n = 97) were randomized to 1 of 3 groups: treatment group A (n = 36), treatment group B (n = 40), and the control group (n = 21). Patients from each group received either of 2 doses of subconjunctival CsA (group A, low-dose CsA; group B, high-dose CsA) or placebo (carrier only) implants at the time of high-risk penetrating keratoplasty. The incidence and degree of corneal neovascularization occurring after penetrating keratoplasty were evaluated in a substudy (LX201-01 study: NCT00447187). A web-based image upload system was developed. Standardized digital slit-lamp pictures were quantitatively and objectively evaluated using CellˆF morphometry software. No statistically significant difference in incidence and degree of corneal neovascularization developing after penetrating keratoplasty was found between treatment groups and placebo group. Mean corneal neovascularization area at week 52 (visit 12) was 2.32±1.79% in treatment group A versus placebo (2.79±2.11%; P = 0.45) and 2.74±2.22% in treatment group B versus placebo (2.79±2.11%; P = 0.94). High-dose subconjunctival CsA implants do not significantly affect corneal neovascularization after high-risk penetrating keratoplasty. This suggests that local CsA has negligible antiangiogenic effects in the human cornea, at least in the transplant setting.

Nox2 is a Mediator of Chronic CsA Nephrotoxicity

We hypothesized that Nox2, the classical phagocytic NADPH oxidase, plays an important role in calcineurin inhibitor (CNI)-induced renal fibrosis. We tested this hypothesis in vitro, in animal and in human studies. Cyclosporine A (CsA) and tacrolimus (TAC) were associated with greater levels of Nox2 mRNA and epithelial to mesenchymal transition (EMT) in NRK52E cells. CsA increased Nox2, α-SMA and phosphorylated-p38MAPK, Smad3 and NFκB proteins. Nox2 upregulation and EMT were inhibited in TGF-β1 knockout cells suggesting that TGF-β1 is required for Nox2 activation. Fisher344 rats treated with high dose CsA showed increased Nox2 in the tubulointerstitium and greater Nox2, α-SMA, phosphorylated Smad3 and nitrotyrosine by immunoblot analyses. Inhibition of Nox2 by coadministration of apocynin or diphenyleneiodonium was associated with reduced fibrogenesis. We validated these findings by treating wild type and Nox2 null (B6.129S-Cybb(Tm1Din)/J) mice with high dose CsA. Western blot analyses confirmed the absence of Nox2 and significantly lower levels of α-SMA and 4-hydroxynonenal (HNE) in CsA-treated knockout mice. These findings were clinically relevant since Nox2 and α-SMA were increased in the tubulointerstitium of kidneys from 15 liver transplant recipients with biopsy-confirmed chronic CsA or TAC nephrotoxicity. In conclusion, specific Nox2 inhibition strategies may improve chronic CNI nephrotoxicity in solid organ transplantation.

Neuroprotective effect of Cyclosporin A on the development of early brain injury in a subarachnoid hemorrhage model: A pilot study

Cyclosporin A (CsA) has been demonstrated to be neuroprotective in ischemic and traumatic brain injuries by inhibiting mitochondrial permeability transition pore (mPTP) opening, thereby maintaining mitochondrial homeostasis and inhibiting pro-apoptotic protein release. The effects of CsA on early brain injury (EBI) after subarachnoid hemorrhage (SAH), however, have not been investigated. This study was designed to explore the effects of CsA on apoptotic signaling pathways and EBI after experimental SAH using four equal groups (n=36) of adult male SD rats, including the sham group, SAH+vehicle group, SAH+CsA2 group, and SAH+CsA10 group. The rat SAH model was induced by injection of 0.3ml non-heparinized arterial blood into the prechiasmatic cistern. In the SAH+CsA2 and SAH+CsA10 groups, a dose of 2mg/kg and 10mg/kg CsA was directly administered by intercarotid injection at 15min and again 24h after SAH induction. Cerebral tissue samples were extracted 48h after SAH. Increased expressions of Cytochrome C, apoptosis-inducing factor (AIF), and cleaved caspase-3 were observed in the cerebral cortex after SAH. Treatment with high dose (10mg/kg) CsA markedly decreased expressions of Cytochrome C, AIF, and cleaved caspase-3, and inhibited apoptosis pathways. Administration of CsA following SAH significantly ameliorated EBI, including cortical apoptosis, brain edema, blood-brain barrier (BBB) impairment, and neurobehavioral deficits. These findings suggest that early administration of CsA may ameliorate EBI and provide neuroprotection in the SAH model through potential mechanisms that include blockage of mPTP opening and inhibition of apoptotic cell death pathways.

Low-dose cyclosporine mediates donor hyporesponsiveness in a fully mismatched rat kidney transplant model

Tolerance induction protocols have been successfully tested in animal models, yet their compatibility with immunosuppressive drugs remains to be fully elucidated. Our own previous data have indicated that cyclosporine A (CsA) affects the balance of effector and regulatory mechanisms with low-dose CsA doses promoting hyporesponsiveness. Here, we present a fully mismatched rat kidney model in which low-dose CsA treatment induces donor hyporesponsiveness to secondary renal allografts. Lewis recipients of DA kidney grafts received low, medium or high doses of CsA×10days. By 30days, the primary transplant was removed and a second transplant of donor origin was engrafted. Following low-dose CsA, but not high-dose CsA treatment of the primary recipient, secondary transplants were accepted long-term in the absence of immunosuppression. Regulatory T-cell function was unimpaired and independent of the CyA dosage. Of note, low-dose CsA significantly reduced alloantibody titers in primary recipients. Adoptive transfer of graft infiltrating cells or splenocytes from hyporesponsive recipients supported long-term acceptance of donor kidney allografts.These results demonstrate a dose-dependent and transferable “pro-tolerogenic” effect of low-dose CsA treatment. This model is of clinical relevance to test the interference of CsA with tolerance induction in the absence of additional immunosuppression.

The role of speckle tracking imaging in the noninvasive detection of acute rejection after heterotopic cardiac transplantation in rats

Objective Acute cardiac allograft rejection continues to be the cause of graft loss and contributes to the morbidity and mortality after cardiac transplantation. Repetitive endomyocardial biopsies are necessary to monitor the eff ects of immunosuppressants after cardiac transplantation. In this study, we investigate whether speckle tracking imaging (STI) is a valuable method in assessing acute cardiac rejection.Methods and resultsHearts from Brown Norway rats or Lewis rats were transplanted into other Brown Norway rats. Isografts and groups of allografts, either untreated or treated with cyclosporine A (CsA) at a low dose (3 mg •kg-1• d-1) or a high dose (10 mg •kg-1• d-1), were compared 7 days after transplantation. Echocardiography-derived left ventricular post wall thickness was increased only in untreated allografts. The left ventricular ejection fraction was signifi cantly lower in the allografts compared with the isografts, but allografts treated without or with low-dose CsA showed similar results .The radial velocity and systolic radial strain rate showed a lower value in untreated allografts than other grafts, but there is no signifi cant diff erence between allografts treated with high- or low-dose CsA and isografts. The circumferential strain and circumferential strain rate were comparable among the 4 groups. However, the radial strain exhibited a clear gradient in these groups (2.8 ± 1.3 in untreated allografts, 5.2 ± 0.9 in allografts treated with lowdose CsA, 6.3 ± 1.8 in allografts treated with high-dose CsA, and 12.7 ± 7.9 in isografts, P < 0.001).Conclusions STI is able to off er a noninvasive method for detecting transplant allograft rejection.

Gender- and dose-related effects of cyclosporin A on hepatic and bone metabolism

Previous data have shown gender-related differences in the skeletal effects of the immunosuppressive drug cyclosporin A (CsA) in rats. To test the hypothesis that the gender-related skeletal effects of CsA are caused by gender-specific metabolism of this drug, we treated aged male and female sham-operated, gonadectomized (GX) as well as sex hormone-supplemented GX rats with 5 mg/kg CsA three times per week for 2 months, and analyzed the bone phenotype as well as the concentrations of CsA and its major metabolites AM1, AM1c, AM9, and AM4N in blood, urine, and liver tissue. CsA treatment induced high turnover osteopenia in males, but not females. Male rats showed several-fold higher CsA and CsA metabolite blood levels compared with females. Renal clearance data revealed that CsA undergoes selective tubular reabsorption in male, but not female rats. However, a mathematical modeling approach demonstrated that the higher CsA blood levels in males were almost exclusively caused by a 6-fold lower hepatic clearance rate compared with females. In addition, we subcutaneously treated female rats with up to 6-fold higher doses of CsA. Similar to males, high dose CsA induced high turnover osteopenia in female rats. Our data show that the gender-related differences in the skeletal effects of CsA are caused by a higher hepatic clearance rate for CsA in female compared to male rats, and not by a differential skeletal response to CsA. Moreover, our study indicates that CsA blood levels of ≤200 ng/ml measured by HPLC do not induce high turnover osteopenia in aged rats.

Evaluating acute rejection after heterotopic cardiac transplantation in rats by speckle tracking imaging

Objective To examine whether speckle tracking imaging(STI) could provide for the assessment of acute cardiac rejection. Methods Hearts from Brown Norway rats or Lewis rats were transplanted into other Brown Norway rats. Isografts and groups of allografts either untreated or treated with cyclosporin A (CsA) at a low dose (3 mg ·kg-1 ·d-1) or high dose (10mg · kg-1 ·d-1) from 1 day before transplantation were compared at posttransplantation day 7. Results Echocardiography-derived left ventricular post wall thickness was increased only in untreated allografts. The left ventricular eject fraction was significant lower in the allografts compared with isograft, but allografts treated without or with low-dose CsA showed similar results. The radial systolic radial strain rate showed a lower value in untreated allografts than other grafts,but there was no significant differences between allograft treated with high- or low-dose CsA and isografts. The circumferential strain and circumferential strain rate was comparable among the 4 groups. However the radial strain exhibited a clear gradient in these groups [(2. 8 ± 1.3)％ in untreated allografts, (5.2 ± 0.9)％ in allografts treated with low-dose CsA, (6.3 ± 1.8 )％ in allografts treated with high-dose CsA,and (12.7 ± 7.9) in isografts, P＜0.001]. The radial strain exhibited a clear correlation with the severity of rejection ( r =-0.812, P＜ 0.0000). Conclusions The radial strain decreased as the severity of rejection worsen. STI offers promise as a noninvasive method for detecting transplant allograft rejection. Key words: Echocardiography; Heart transplantation; Graft rejection; Speckle tracking imaging

Cyclosporin A Inhibits Albumin Synthesis in Huh7 Cells

Background/AimsHypoalbuminemia occurs frequently in renal transplant recipients immediately after renal transplantation. We studied the regulation of hepatic albumin synthesis by cyclosporin A (CsA) in Huh7 cells.MethodsHuh7 cells were incubated with various concentrations of CsA for 4, 8, 16, and 24 hours. Albumin was measured in Huh7 cell-conditioned medium by sandwich enzyme-linked immunosorbent assay and Western blot. Albumin mRNA expression was analyzed by Northern blotting in CsA-treated cells.ResultsCsA (10-7-10-4 M) inhibited albumin synthesis in Huh7 cells in a dose- dependent manner. A Western blot analysis for albumin in the conditioned medium released from CsA-treated (10-7-10-5 M) cells also showed significant inhibition of albumin synthesis in a dose-dependent manner. Vehicle (olive oil) did not affect albumin synthesis. In contrast, a Northern blot analysis revealed no inhibition of albumin mRNA expression by CsA at any time point from 1-24 hours, indicating that the inhibition of albumin synthesis occurred at the translational level.ConclusionsOur results suggest that inhibition of hepatic albumin synthesis by high dose CsA contributes to the hypoalbuminemia in renal transplant recipients.

GVHD prophylaxis using low‐dose cyclosporine improves survival in leukaemic recipients of HLA‐identical sibling transplants

Graft-versus-host disease (GVHD) prophylaxis of short duration (6 months) with low-dose cyclosporine A (CsA) starting at 1 mg/kg per day i.v. and four doses of methotrexate (MTX) were given to 171 consecutive leukaemic recipients of HLA-identical sibling transplants. In contrast, apart from MTX, retrospective controls received high-dose CsA, starting at 5-7.5 mg/kg per day i.v. and discontinued 1 yr post-transplant. In the low-dose CsA group, the probability of acute GVHD grades I-II (70% vs. 53%, P < 0.01), and chronic GVHD were increased (58% vs. 25%, P < 0.01), whereas the incidences of acute GVHD grades III-IV (9% vs. 5%, P = 0.62), and non-relapse mortality (20% vs. 22%, P = 0.58) were similar. Moreover, the probability of relapse was decreased (31% vs. 54%, P < 0.01), and both relapse-free (56% vs. 38%, P = 0.04) and overall survival (61% vs. 40%, P = 0.04) were markedly improved using the low-dose CsA regimen. In multivariate analyses, low-dose CsA was strongly associated with chronic GVHD [relative hazard (RH) 2.56, P < 0.01], which decreased the risk of relapse (RH 0.46, P < 0.01) and improved the probability of survival (RH 1.84, P < 0.01). In conclusion, a low-dose CsA regimen in leukaemic recipients of HLA-identical sibling transplants increases the rate of chronic GVHD, which seems to attenuate the risk of relapse, thereby improving patient survival owing to enhanced graft-versus-leukaemia effect.

Cyclosporin A is superior to cyclophosphamide in children with steroid-resistant nephrotic syndrome-a randomized controlled multicentre trial by the Arbeitsgemeinschaft für Pädiatrische Nephrologie.

First line immunosuppressive treatment in steroid-resistant nephrotic syndrome in children is still open to discussion. We conducted a controlled multicentre randomized open label trial to test the efficacy and safety of cyclosporin A (CSA) versus cyclophosphamide pulses (CPH) in the initial therapy of children with newly diagnosed primary steroid-resistant nephrotic syndrome and histologically proven minimal change disease, focal segmental glomerulosclerosis or mesangial hypercellularity. Patients in the CSA group (n = 15) were initially treated with 150 mg/m(2) CSA orally to achieve trough levels of 120-180 ng/ml, while patients in the CPH group (n = 17) received CPH pulses (500 mg/m(2) per month intravenous). All patients were on alternate prednisone therapy. Patients with proteinuria >40 mg/m(2) per hour at 12 weeks of therapy were allocated to a non-responder protocol with high-dose CSA therapy or methylprednisolone pulses. At week 12, nine of the 15 (60%) CSA patients showed at least partial remission, evidences by a reduction of proteinuria <40 mg/h per m(2). In contrast, three of the 17 (17%) CPH patients responded (p < 0.05, intention-to-treat). Given these results, the study was stopped, in accordance with the protocol. After 24 weeks, complete remission was reached by two of the 15 (13%) CSA and one of the 17 (5%) CPH patients (p = n.s.). Partial remission was achieved by seven of the 15 (46%) CSA and two of the 15 (11%) CPH patients (p <0.05). Five patients in the CSA group and 14 patients in the CPH group were withdrawn from the study, most of them during the non-responder protocol. The number of adverse events was comparable between both groups. We conclude that CSA is more effective than CPH in inducing at least partial remission in steroid-resistant nephrotic syndrome in children.

SEX DIFFERENCES IN RESPONSE TO CYCLOSPORINE IMMUNOSUPPRESSION IN EXPERIMENTAL KIDNEY TRANSPLANTATION

1. Female donors and recipients have increased risk of acute rejection and subsequent chronic allograft nephropathy (CAN), especially when cyclosporine A (CsA) is used. Decreased renal nitric oxide (NO) production is associated with chronic kidney disease. In the present study, we investigated the impact of gender, CsA dose and renal NO synthase (NOS) on CAN. 2. Kidneys from male and female F344 rats were transplanted into same-sex Lewis allograft or F344 isograft recipients and recipient rats were treated with 1.5 or 3 mg/kg per day CsA for 10 days. Grafts were removed at 22 weeks post-transplantation. Normal two-kidney F344 rats were investigated as age-matched controls. 3. Low-dose CsA was associated with accelerated CAN in female rats compared with male rats; however, with high-dose CsA, allograft females had similar pathology/function to allograft males. Isograft females (similar to isograft males) had no graft failure and only slightly, albeit significantly, greater injury than age-matched controls. Isograft females had higher renal cortical neuronal (n) NOS but lower medullary endothelial (e) NOS than isograft males. There was no difference in renal eNOS and nNOS between allograft groups. 4. In conclusion, 1.5 mg/kg per day CsA is not sufficient to prevent early graft loss in females. When the dose of CsA is doubled, allograft females and males have similar post-transplant survival. Renal NOS expression was unremarkable in any transplant group.

Influence of cyclosporine A on the mRNA and protein expression of major histocompatibility complexes I and II: experiment with rabbits

To investigate the influence of cyclosporine A (CsA) on the mRNA and protein expression of major histocompatibility (MHC) I and II. 14 New Zealand white rabbits were randomly divided into three groups to be injected intramuscularly 5 mg/kg once a day for 30 days (low dose CsA group, n = 5), 20 mg/kg (high dose CsA group, n = 5), or normal saline (control group, n = 4). Peripheral blood sample was collected every 5 days, monouclear cells were isolated, total RNA was extracted, and 30 days later the rabbits were killed and their livers, spleens, kidneys, and thyroid glands were taken out, total RNA was extracted, RT-PCR and Western blotting were used to examine the mRNA and protein expression of MHCI and MHCII. The peripheral blood monouclear cell MHCI and MHCII mRNA expression levels on day 10 of the low dose CsA group were 1.78 +/- 0.15 and 7.30 +/- 0.51, both significantly higher than those before the injection of CsA (0.23 +/- 0.04 and 2.77 +/- 0.25, both P < 0.01); the MHCI and MHCII protein expression levels were 11.32 +/- 1.45 and 22.31 +/- 1.91, both significantly higher than those before the injection (2.31 +/- 0.14 and 12.52 +/- 1.23, both P < 0.01). In high dose CsA group, the MHCI and MHCII mRNA expression levels on day 10 were 4.72 +/- 0.28 and 14.20 +/- 2.58, both significantly higher than those before the injection (0.24 +/- 0.06 and 2.56 +/- 0.33, both P < 0.01); the protein expression levels were 13.81 +/- 1.38 and 24.22 +/- 2.88, both significantly higher than those before the injection (2.42 +/- 0.06 and 12.51 +/- 1.31, both P < 0.01). Since the day 15, the levels of MHCI and MHCII mRNA and protein expression levels began decrease gradually until the levels before the injection. By the end of the experiment there was no significant difference in the MHCI and MHCII mRNA and protein expression levels of the liver, spleen, kidney, and thymus among the 3 groups. Cyclosporine can increase the host MHCI and MHCII expression for a period of time.

Activation of PPARγ enhances in vitro the immunosuppressive effect of cyclosporine on T lymphocytes

Background Peroxisome Proliferator-Activated Receptor γ (PPARγ) is a nuclear receptor that regulates the transcription of genes associated with lipid and glucose metabolism. Recently, it has been shown that PPARγ modulates the activity of T cells, resulting in inhibition of T cell proliferation and IL-2 release. In this study we investigated whether the PPARγ ligand rosiglitazone (R) enhances in vitro the immunosuppressive effects of cyclosporine A (CsA). Methods CD4 + T cells isolated from peripheral blood mononuclear cells of healthy donors were activated either with mitogens or by one-way mixed lymphocyte reaction. The activated T cells were treated with (1) CsA at low and high concentration (50, 150 ng/ml); (2) R (20 μM); (3) R (20 μM) in combination with CsA at low concentration (50 ng/ml). We studied the effects of the various treatments on cell proliferation (incorporation of [ 3H] thymidine), the cell-cycle phases (FACS analysis), IL-2 release (ELISA), and IL-2 receptor (CD25) expression (FACS analysis). Results R used alone reduced T cell proliferation and CD25 expression. Low-dose CsA combined with R was significantly more powerful than either high-dose CsA alone or R alone in suppressing IL-2 release, arresting the T cell cycle, and blocking the growth of activated T cells. Conclusion PPARγ ligand R potentiates in vitro the inhibitory action of CsA on activated T helper cells. The combined use of PPARγ ligands and low-dose CsA represents a rationale therapeutic approach aimed to prevent CsA nephrotoxicity while maintaining adequate immunosuppression.

Analysis of Allogeneic and Syngeneic Bone Marrow Stromal Cell Graft Survival in the Spinal Cord

Bone marrow stromal cells (MSC) are attractive candidates for developing cell therapies for central nervous system (CNS) disorders. They can be easily obtained, expanded in culture, and promote modest functional recovery following transplantation into animal models of injured or degenerative CNS. While syngeneic MSC grafts can be used efficiently, achieving long-term survival of allogeneic MSC grafts has been a challenge. We hypothesize that improved graft survival will enhance the functional recovery promoted by MSC. To improve MSC graft survival, we tested two dosages of the immune suppressant cyclosporin A (CsA) in an allogeneic model. Syngeneic transplantation of MSC where cells survive well without immune suppression was used as a control. Sprague-Dawley rats treated with standard dose (n = 12) or high-dose (n = 12) CsA served as allogeneic hosts; Fisher 344 rats (n = 12) served as syngeneic hosts. MSC were derived from transgenic Fisher 344 rats expressing human placental alkaline phosphatase and were grafted into cervical spinal cord. Animals treated with standard dose CsA showed significant decreases in allograft size 4 weeks posttransplantation; high CsA doses yielded significantly better graft survival 4 and 8 weeks posttransplantation compared to standard CsA. As expected, syngeneic MSC transplants showed good graft survival after 4 and 8 weeks. To investigate MSC graft elimination, we analyzed immune cell infiltration and cell death. Macrophage infiltration was high after 1 week in all groups. After 4 weeks, high-dose CsA and syngeneic animals showed significant reductions in macrophages at the graft site. Few T lymphocytes were detected in any group at each time point. Cell death occurred throughout the study; however, little apoptotic activity was detected. Histochemical analysis revealed no evidence of neural differentiation. These results indicate that allogeneic transplantation with appropriate immune suppression permits long-term survival of MSC; thus, both allogeneic and syngeneic strategies could be utilized in devising novel therapies for CNS injury.