Cyclosporin A Immunosuppression Research Articles

Centrifugally concentric ring-patterned drug-loaded polymeric coating as an intraocular lens surface modification for efficient prevention of posterior capsular opacification

Posterior capsular opacification (PCO) is the main postoperative complication after intraocular lens (IOL) implantation in cataract surgery, because of the proliferation of the residual lens epithelial cells (LECs) in the lens capsule. Drug-eluting IOLs, aimed to develop an in situ drug delivery device, are the promising concept in recent years. As IOLs are optical devices other than implants, the feasibility and applicability remain a challenge for drug-eluting coatings. In this investigation, a centrifugally concentric ring-patterned drug-loaded poly(lactide-co-glycolic acid) (PLGA) coating was designed and fabricated by the spin coating technique. The concentric ring-patterned morphologies and the drug loading and release properties were carefully investigated, and the spin coating parameters were optimized. A concentric annular coating with a thin center and thick periphery was obtained, which was particularly suitable for the surface modification of IOLs, as the visual pathway of the intraocular light transmission greatly requires good light transmittance of the IOLs. IOLs with the immunosuppressant cyclosporin A (CsA)-loaded coating (CsA @ PLGA) modification were then fabricated for PCO prevention. The in vitro LECs culture results showed that the CsA @ PLGA coating-modified IOLs significantly inhibited cell proliferation and induced cell death. Western blot analysis showed that the efficient cell inhibition behavior of CsA was due to the autophagy-mediated cell death pathway. The in vivo intraocular implantation results confirmed the desired PCO inhibition effect. Thus, the centrifugally concentric ring-patterned drug-loaded PLGA coating obtained by the spin coating technique provides a simple yet effective alternative of IOL modification for PCO prevention. Statement of Significance• Concentric ring-patterned polymer coating, specifically for drug-eluting IOL fabrication, was developed by the spin coating technique.• The immunosuppressant CsA inhibited LEC proliferation through the autophagy-mediated cell death pathway.• Concentric ring-patterned CsA-eluting IOLs exhibited reliable in vivo PCO prevention.• The drug-eluting IOLs fabricated by the simple and economical spin coating technique have a great potential in clinical translation.

Cyclophilins A and B oppositely regulate renal tubular epithelial cell phenotype

Restoration of kidney tubular epithelium following sublethal injury sequentially involves partial epithelial–mesenchymal transition (pEMT), proliferation, and further redifferentiation into specialized tubule epithelial cells (TECs). Because the immunosuppressant cyclosporine-A produces pEMT in TECs and inhibits the peptidyl-prolyl isomerase (PPIase) activity of cyclophilin (Cyp) proteins, we hypothesized that cyclophilins could regulate TEC phenotype. Here we demonstrate that in cultured TECs, CypA silencing triggers loss of epithelial features and enhances transforming growth factor β (TGFβ)-induced EMT in association with upregulation of epithelial repressors Slug and Snail. This pro-epithelial action of CypA relies on its PPIase activity. By contrast, CypB emerges as an epithelial repressor, because CypB silencing promotes epithelial differentiation, prevents TGFβ-induced EMT, and induces tubular structures in 3D cultures. In addition, in the kidneys of CypB knockout mice subjected to unilateral ureteral obstruction, inflammatory and pro-fibrotic events were attenuated. CypB silencing/knockout leads to Slug, but not Snail, downregulation. CypB support of Slug expression depends on its endoplasmic reticulum location, where it interacts with calreticulin, a calcium-buffering chaperone related to Slug expression. As CypB silencing reduces ionomycin-induced calcium release and Slug upregulation, we suggest that Slug expression may rely on CypB modulation of calreticulin-dependent calcium signaling. In conclusion, this work uncovers new roles for CypA and CypB in modulating TEC plasticity and identifies CypB as a druggable target potentially relevant in promoting kidney repair.

Adenovirus Biodistribution is Modified in Sensitive Animals Compared to Naïve Animals.

Pre-existing immune response against adenovirus could diminish transgene expression efficiency when Ad is employed in humans as gene therapy vector. We previously used Ad-hΔuPA (Recombinant adenovirus expressing human urokinase-type plasminogen activator) as antifibrotic gene therapy in cirrhosis models and demonstrated its effectiveness. As a further clinical approach, transient Cyclosporine A (CsA) immunosuppression was induced in cirrhotic animals to determine whether Ad-hΔuPA administration retained efficacy. Adenovirus sensitization was achieved by systemic administration of non-therapeutic Ad-βGal (Recombinant adenovirus expressing beta-galactosidase) after 4weeks of intraperitoneal carbon tetrachloride (CCl4) regimen. Cirrhosis induction continued up to 8weeks. At the end of CCl4 intoxication, immunosuppression was achieved with three CsA doses (40mg/kg) as follows: 24h before administration of Ad-hΔuPA, at the moment of Ad-hΔuPA injection and finally, 24h after Ad-hΔuPA inoculation. At 2 and 72h after Ad-hΔuPA injection, animals were sacrificed. Liver, spleen, lung, kidney, heart, brain, and testis were analyzed for Ad-biodistribution and transgene expression. In naïve animals, Ad-hΔuPA genomes prevailed in liver and spleen, while Ad-sensitized rats showed Ad genomes also in their kidney and heart. Cirrhosis and Ad preimmunization status notably diminished transgene liver expression compared to healthy livers. CsA immunosuppression in cirrhotic animals has no effect on Ad-hΔuPA biodistribution, but increments survival.

Synergism of a natural plant product, oleanolic acid with calcineurin inhibitor in prolonging islet allograft survival

BackgroundOleanolic acid (OA) is a natural plant-derived triterpenoid with potent anti-inflammatory properties. Since inflammatory cytokines released following islet transplantation hinders engraftment and long-term function, we determined the synergistic ability of OA to with Cyclosporine-A (CSA), a calcineurin inhibitor in improving islet allograft's function and survival. MethodsC57BL/6 mice were rendered diabetic using streptozotocin (200mg/kg). BALB/c islets were transplanted under the kidney capsule alone (control) or along with administration of OA alone, CSA alone or a combination of both OA and CSA (OA+CSA). T-cell infiltration was analyzed by immunohistochemistry; cytokine concentration was analyzed by Luminex; T-cell cytokine phenotype was analyzed by ELISpot; and alloimmune response was analyzed by flow cytometry. ResultsOA+CSA markedly prolonged islet allograft survival compared to controls (37±5days vs. 8±3days). A significant decrease of CD4+ (34±9 vs. 154±42cells/hpf) and CD8+ T-cellular (46±22 vs. 224±51cells/hpf, p<0.0001) infiltration into the graft in OA+CSA treated mice compared to controls. A significant decrease in T cell infiltration was demonstrated in the OA+CSA cohort over either compound application individually. An increase in anti-inflammatory molecules, IL-10 (2.4-fold) and vascular endothelial growth factor (1.6-fold), along with decreased pro-inflammatory cytokines IFN-γ, IL-1β (1.3–2.4-fold) and IL-17 (3.2-fold) was demonstrated. OA+CSA also significantly decreased the frequency of allo-specific T-cell responses. Development of antibodies against donor antigens was also delayed (39 vs 22days; p<0.05) in the OA+CSA cohort over administration of either agent individually. ConclusionsOA and CSA exert synergistic effect towards enhancing islet allograft survival and function. This synergistic effect resulted in markedly reduced graft infiltrating cells with attenuation of inflammatory cytokine milieu leading to impairment of both cellular and humoral alloimmune responses. Therefore, novel therapeutic approaches involving combination of OA with calcineurin-inhibitor based immunosuppressant CSA will produce potential beneficial outcomes in clinical islet transplantation.

Crosstalk between Shh and TGF-β Signaling in Cyclosporine-Enhanced Cell Proliferation in Human Gingival Fibroblasts

BackgroundImmunosuppressant cyclosporine-A induces gingival hyperplasia, which is characterized by increased fibroblast proliferation and overproduction of extracellular matrix components and regulated by transforming growth factor-beta (TGF-β). The TGF-β and Sonic hedgehog (Shh) pathways both mediate cell proliferation. Crosstalk between these pathways in cancer has recently been proposed, but the hierarchical pattern of this crosstalk remains unclear. In normal fibroblasts, a TGF-β-stimulating Shh pattern was observed in induced fibrosis. However, Shh pathway involvement in cyclosporine-enhanced gingival proliferation and the existence of crosstalk with the TGF-β pathway remain unclear.Methodology/Principal FindingsCyclosporine enhanced mRNA and protein levels of TGF-β and Shh in human gingival fibroblasts (RT-PCR and western blotting). A TGF-β pathway inhibitor mitigated cyclosporine-enhanced cell proliferation and an Shh pathway inhibitor attenuated cyclosporine-enhanced proliferation in fibroblasts (MTS assay and/or RT-PCR of PCNA). Exogenous TGF-β increased Shh expression; however, exogenous Shh did not alter TGF-β expression. The TGF-β pathway inhibitor mitigated cyclosporine-upregulated Shh expression, but the Shh pathway inhibitor did not alter cyclosporine-upregulated TGF-β expression.Conclusions/SignificanceThe TGF-β and Shh pathways mediate cyclosporine-enhanced gingival fibroblast proliferation. Exogenous TGF-β increased Shh expression, and inhibition of TGF-β signaling abrogated the cyclosporine-induced upregulation of Shh expression; however, TGF-β expression appeared unchanged by enhanced or inhibited Shh signaling. This is the first study demonstrating the role of Shh in cyclosporine-enhanced gingival cell proliferation; moreover, it defines a hierarchical crosstalk pattern in which TGF-β regulates Shh in gingival fibroblasts. Understanding the regulation of cyclosporine-related Shh and TGF-β signaling and crosstalk in gingival overgrowth will clarify the mechanism of cyclosporine-induced gingival enlargement and help develop targeted therapeutics for blocking these pathways, which can be applied in pre-clinical and clinical settings.

Kidney Transplantation Down-Regulates Expression of Organic Cation Transporters, Which Translocate β-Blockers and Fluoroquinolones

Kidney transplanted patients are often treated with immunosuppressive, antihypertensive, and antibiotic drugs such as cyclosporine A (CsA), β-blockers, and fluoroquinolones, respectively. Organic cation transporters (OCT) expressed in the basolateral membrane of proximal tubules represent an important drug excretion route. In this work, the renal expression of OCT after syngeneic and allogeneic kidney transplantation in rats with or without CsA immunosuppression was studied. Moreover, the interactions of CsA, β-blockers (pindolol/atenolol), and fluoroquinolones (ofloxacin/norfloxacin) with rOCT1, rOCT2, hOCT1, and hOCT2 in stably transfected HEK293-cells were studied. Kidney transplantation was associated with reduced expression of rOCT1, while rOCT2 showed only reduced expression after allogeneic transplantation. All drugs interacted subtype- and species-dependently with OCT. However, only atenolol, pindolol, and ofloxacin were transported by hOCT2, the main OCT in human kidneys. While CsA is not an OCT substrate, it exerts a short-term effect on OCT activity, changing their affinity for some substrates. In conclusion, appropriate drug dosing in transplanted patients is difficult partly because OCT are down-regulated and because concomitant CsA treatment may influence the affinity of the transporters. Moreover, drug-drug competition at the transporter can also alter drug excretion rate.

Placebo-induced immunosuppression in humans: Role of learning and expectation

We analyzed if behaviorally conditioned immunosuppression is affected by the number of re-expositions to the conditioned stimulus (CS) during evocation and whether an immunosuppression can be induced through expectation. During the acquisition of the conditioning procedure, subjects received the immunosuppressant cyclosporine-A (unconditioned stimulus/US) ( n = 42) or a placebo ( n = 23) with a green-colored novel tasting drink (CS) four times. Five days later, participants were re-exposed to the CS paired with placebo pills either four times ( n = 32) or only once ( n = 19). Interleukin (IL)-2 production of anti-CD3 stimulated PBMCs was analyzed before and after the acquisition and evocation. Subjects re-exposed four times to the CS during evocation, showed a behaviorally conditioned immunosuppression (significantly decreased IL-2 production). A single re-exposition to the CS did not induce an immunosuppressive effect. Furthermore, we investigated whether an immunosuppression may be induced through expectation. Participants were told to have different probabilities ( n = 8–9 per condition) of receiving cyclosporine-A but in fact received placebo. We analyzed IL-2 production before the first placebo intake and two hours after the last intake. Expectation did not affect IL-2 production in any group. In conclusion, immunosuppressive effects can be induced through behavioral conditioning (after multiple re-exposures to the CS) but not through expectation.

Overview of Gingival Overgrowth in Transplant Patients

Patients with gingival overgrowth are easily seen in dental clinics. Cyclosporin-A (CsA), a widely prescribed immunosuppressant induces gingival overgrowth in up to 35% of patients with medical history of organ transplantation. The immunosuppressant CsA can transform genetic expression of gingival fibroblasts, resulting in gingival overgrowth. Meticulous plaque control is recommended for treatment of gingival overgrowth. Substitution of the drug or surgical procedures such as gingivectomy and periodontal flaps should be considered after re-evaluation. Azithromycin is often recommended as a supplementary drug to reduce this side effect. Recent studies show that tacrolimus can be a more economic, efficient and safe substitute for CsA.

Anemia is not predictive of sustained virological response in liver transplant recipients with hepatitis C virus who are treated with pegylated interferon and ribavirin

In the immunocompetent setting, antiviral therapy-related anemia has recently been shown to be associated with a sustained virological response (SVR). Our goal was to assess whether this is also true for liver transplantation (LT). We included 160 LT patients with recurrent hepatitis C virus (HCV) who were treated with pegylated interferon and ribavirin (RBV) between 2002 and 2010; 76% of the patients were men, the median age of the patients was 56 years (range = 33-75 years), 63% had advanced fibrosis, and 86% were infected with HCV genotype 1a or 1b. The baseline immunosuppression was tacrolimus in 56% of the patients. Mycophenolate mofetil (MMF) was used in 15%. Anemia was defined as a hemoglobin (Hb) level < 10 g/dL. Significant anemia was present when the Hb decline was >5 g/dL. Anemia and significant anemia developed in 67% and 41% of the patients, respectively. Erythropoietin was used in 60%. Factors independently associated with significant anemia included low estimated creatinine clearance [relative risk (RR) = 0.951, 95% confidence interval (CI) = 0.925-0.978, P = 0.0001], a longer time from LT to therapy (RR = 1.001, 95% CI = 1.000-1.001, P = 0.002), high baseline viremia (RR = 3.2, 95% CI = 1.3-8.1, P = 0.01), cyclosporine A (CSA)-based immunosuppression (RR: 3.472, 95% CI: 1.386-8.695; P = 0.008), and the use of MMF (RR: 5.346, 95% CI: 1.398-20.447; P = 0.014). An SVR occurred in 43% of the patients; the factors associated with an SVR included baseline variables (younger recipient age, younger donor age, infections with non-1 HCV genotypes, body mass index, and mild fibrosis) and on-treatment factors related to adherence or viral kinetics. Anemia resulted in RBV dose reductions but was not associated with the virological response at any time. In conclusion, anemia is a very frequent complication in LT patients during antiviral therapy and is associated with increased RBV dose reduction but not with an SVR. Predictors of anemia include MMF or CSA immunosuppression, high viremia, and renal insufficiency.

The effects of Cyclosporine A and azathioprine on human T cells activated by different costimulatory signals

Immunosuppression is an important treatment modality in transplantation and human diseases that are associated with aberrant T cell activation. There are considerable differences regarding the cellular processes targeted by the immunosuppressive drugs that are in clinical use. Drugs like azathioprine (Aza) mainly act by halting proliferation of fast dividing cells, whereas others like cyclosporine A (CsA) specifically target signaling pathways in T cells. Since the outcome of T cell responses critically depends on the quality and strength of costimulatory signals, this study has addressed the interplay between costimulation and the immunosuppressive agents CsA and Aza during the in vitro activation of human T cells. We used an experimental system that allows analyzing T cells activated in the presence of selected costimulatory ligands to study T cells stimulated via CD28, CD2, LFA-1, ICOS or 4-1BB. The mean inhibitory concentrations (IC50) for Aza and CsA were determined for the proliferation of T cells receiving different costimulatory signals as well as for T cells activated in the absence of costimulation. CD28 signals but not costimulation via CD2, 4-1BB, ICOS or LFA-1 greatly increased the IC50 for CsA. By contrast, the inhibitory effects of Aza were not influenced by T cell costimulatory signals. Our results might have implications for combining standard immunosuppressive drugs with CTLA-4Ig fusion proteins, which act by blocking CD28 costimulation.

Pure red cell aplasia associated with hemolytic anemia refractory to standard measures and resolved by rituximab in an elderly patient

Acquired pure red cell aplasia (PRCA) simultaneously associated with autoimmune hemolytic anemia (AIHA) has been very rarely described so far [1, 2]. We describe the case of an elderly woman, who was diagnosed as having idiopathic PRCA associated with AIHA which was unresponsive to two lines of immunosuppressive treatment and was then resolved by rituximab given as salvage therapy. A 68-year-old woman kept under our attention because of severe malaise, pallor and fatigue. Her past medical history was unremarkable; in particular, she denied any personal or familiar history of hereditary hemolytic anemia or autoimmune diseases. A past blood count had shown normal hemoglobin. Physical examination at admission was unremarkable, with the exception of pallor and mild jaundice; the patient did not present either spleen or liver enlargement. A comprehensive laboratory evaluation revealed no abnormalities other than those related to anemia and hemolysis. Indeed, severe normochromic and normocytic anemia (hemoglobin, Hb = 2.7 g/dl) was revealed. Reticulocytes were not detectable; white blood cell and platelet counts were normal. Increased indirect serum bilirubin, urinary urobilinogen and serum lactate dehydrogenase were present; serum haptoglobin was undetectable. Direct and indirect Coombs tests were positive. A warm-reactive antibody of the IgG isotype was eluted from the red blood cells (RBC). Serologic tests for parvovirus B19, HIV, Hepatitis virus, human cytomegalovirus, and Epstein-Barr virus were negative. Moreover, no clinical and/or laboratory signs of associated autoimmune disease were detected. Chest X-ray and total body CT scan showed normal findings. On the blood film, spherocytes and polychromasia were found. A bone marrow (BM) aspirate and a trephine biopsy demonstrated normal representation of myeloid and megakaryocytic precursors, but nearly absent erythroid precursors. Based on these findings, a diagnosis of AIHA combined to PRCA was made. Given the symptomatic anemia and the very low Hb levels, the patient received repeated administration of RBC concentrates. Methylprednisolone 2 mg/kg was given for 4 weeks without any benefit; equally ineffective was the concomitant treatment with cyclosporine-A (CSA) during the following 4 weeks. Therefore, in the light of reported experiences concerning the treatment of AIHA [3] and PRCA [2], the patient was offered rituximab as salvage therapy for which she gave her informed consent. Rituximab was administered intravenously at the dose of 375 mg/m as a 4-h infusion, once weekly for a total of four doses, without any adverse reaction or side effects (July 2007). Progressive increase of Hb levels and achievement of transfusion independence followed the rise in reticulocytes after the fourth dose of rituximab. Serum hemolytic parameters normalized, as well as the serologic finding of an auto-antibody also disappeared. A BM aspirate demonstrated no abnormal findings and, in particular, the full recovery of the erythroid matrix. The patient, 10 months after rituximab therapy, shows normal Hb and reticulocyte levels, and she is no longer received immunosuppressive therapy. Treatment of PRCA and of AIHA usually employs immunosuppressive drugs, mainly steroids and CSA, or immunomodulating agents [1]. However, conventional immunosuppressive treatments are often unsatisfactory for which rituximab has been successfully used as salvage therapy in the pediatric setting [2]. Our L. Scaramucci (&) P. Niscola M. Ales M. Giovannini A. Tendas L. Cupelli A. Siniscalchi D. Piccioni T. Dentamaro A. Perrotti P. de Fabritiis Hematology Unit, Sant’Eugenio Hospital, Piazzale dell’Umanesimo 10, 00144 Rome, Italy e-mail: scaramucci.laura@gmail.com

CDNA microarray analysis of cyclosporin A (CsA)-treated human peripheral blood mononuclear cells reveal modulation of genes associated with apoptosis, cell-cycle regulation and DNA repair

Cyclosporin A (CsA) is a potent immunosuppressant that has been extensively used to attenuate patient immune response following organ transplantation. The molecular biological mechanism of CsA has been extensively investigated in human T cells, and it has been shown to involve modulation of the intracellular calcineurin pathway. However, it is plausible that this chemical immunosuppressant certainly up- or down-regulate many other biochemical pathways of immune cells. In the present study, we used the cDNA microarray method to characterize the gene expression profile of human peripheral blood mononuclear cells (PBMC) treated in vitro with CsA and controls. The CsA treated PBMC displayed statistically significant induction of genes involved in the control of cell-cycle regulation (TRRAP), apoptosis/DNA repair (PRKDC, MAEA, TIA1), DNA metabolism/response to DNA damage stimulus (PRKDC, FEN1), transcription (NR4A2, THRA) and cell proliferation (FEN1, BIN1), whose data have permitted identification of target genes involved in CsA immunosuppression.

Acute and Chronic Vascular Rejection in Nonhuman Primate Kidney Transplantation

A nonhuman primate (NHP) study was designed to evaluate in nonlife-supporting kidney allografts the progression from acute rejection with transplant endarteritis (TXA) to chronic rejection (CR) with sclerosing vasculopathy. Group G1 (n = 6) received high cyclosporine A (CsA) immunosuppression and showed neither TXA nor CR during 90 days post-transplantation. Group G2 (n = 6) received suboptimal CsA immunosuppression and showed severe TXA with graft loss within 46 days (median). Arterial intimal changes included infiltration of macrophages and T lymphocytes (CD3, CD4, CD8) with few myofibroblasts, abundant fibronectin/collagen IV, scant collagens I/III, high rate of cellular proliferation and no C4d accumulation along peritubular capillaries. Group G3 (n = 12) received suboptimal CsA and anti-rejection therapy (rabbit ATG + methylprednisolone + CsA) of TXA. Animals developed CR and lost grafts within 65 days (median). As compared to G2, the arterial intimal changes showed less macrophages and T lymphocytes, an increased number of myofibroblasts, abundant fibronectin/collagen IV and scar collagens I/III, C4d deposition along capillaries in 60% of animals and transplant glomerulopathy in 80% of animals. In conclusion, CR is an immune stimulated process initiated during TXA with the accumulation and proliferation of myofibroblasts, and progressive deposition of collagens in the intima. Our experimental design appears well suited to study events leading to CR.

Role of the CCAAT/Enhancer-binding Protein NFATc2 Transcription Factor Cascade in the Induction of Secretory Phospholipase A2

Inflammatory cytokines such as interleukin-1 and tumor necrosis factor-alpha modulate a transcription factor cascade in the liver to induce and sustain an acute and systemic defense against foreign entities. The transcription factors involved include NF-kappaB, STAT, and CCAAT/enhancer-binding protein (C/EBP). Whether the NFAT group of transcription factors (which was first characterized as playing an important role in cytokine gene expression in the adaptive response in immune cells) participates in the acute-phase response in hepatocytes is not known. Here, we have investigated whether NFAT is part of the transcription factor cascade in hepatocytes during inflammatory stress. We report that interleukin-1 or tumor necrosis factor-alpha increases expression of and activates NFATc2. C/EBP-mediated NFATc2 induction is temporally required for expression of type IIA secretory phospholipase A2. NFATc2 is also required for expression of phospholipase D1 and the calcium-binding protein S100A3. Thus, a C/EBP-NFATc2 transcription factor cascade provides an additional means to modulate the acute-phase response upon stimulation with inflammatory cytokines.

Neural stem cell grafts reduce the extent of neuronal damage in a mouse model of global ischaemia

The therapeutic potential of neural stem cell transplantation has been well demonstrated in many models of focal brain damage. However, few studies have sought to determine whether neural stem cells are therapeutic in models of diffuse brain injury, such as observed in Alzheimer's disease and global ischaemia. The present study investigated the effects of transplanted MHP36 neural stem cells on the extent of ischaemic damage in a mouse model of global ischaemia and the effects of the immunosuppressive agent cyclosporin A (CsA). C57Bl/6J mice received an intrastriatal graft of MHP36 neural stem cells 3 days after selective neuronal damage had been induced by global ischaemia. The experimental group was subdivided into CsA or saline controls. We discovered that grafts of MHP36 neural stem cells were able to differentiate into neurons and reduce the extent of ischaemic neuronal damage. This reduction was particularly apparent at 4 week post-transplantation and is independent of CsA immunosuppression. MHP36 cells survived robustly in host ischaemic brain and migrated away from the injection tract towards the caudate nucleus and corpus callosum. Although MHP36 grafts were associated with an acute inflammatory response from reactive astrocytes and microglia at 1 week post-transplantation, this decreased markedly by 4 weeks post-transplantation even in the absence of CsA immunosuppression. This is the first study showing a therapeutic benefit of neural stem cells in a highly diffuse brain injury, further highlighting the possibilities of stem cell transplantation for all types of neurodegenerative disease.

Aortic allograft vasculopathy is mediated by CD8 + T cells in Cyclosporin A immunosuppressed mice

We investigated the role of CD4 + T cells and CD8 + T cells in mediating allograft vasculopathy in Cyclosporin A (CyA) immunosuppressed mice. We first established that a dose of 50 mg/kg/d CyA was required to prevent acute rejection in C57BL/6 mice. CyA given at 50 mg/kg/d did not prevent allograft vasculopathy in either cardiac or aortic transplants in these mice. Using CD4 −/− and CD8 −/− mice, we established that CyA immunosuppression at this dose was only effective at preventing allograft vasculopathy in mice lacking CD8 + T cells. This implicates CD8 + T cells in the development of AV in situations of clinical cardiac transplantation where CyA is still the mainstay of immunosuppressive therapy. We confirmed the important role for CD8 + T cells in AV in the face of CyA immunosuppression by allopriming mice in the presence of CyA and transferring alloprimed T cells into RAG1 −/− immunodeficient mice. The RAG1 −/− mice were also treated with CyA. In this situation (CyA present during the allopriming and in the recipient), only primed CD8 + T cells could mediate AV, primed CD4 + T cells could not. Alloprimed CD8 + T cells raised in the presence of CyA exhibited markedly reduced direct recognition responses (as measured by MLR) and effector responses (as measured by cytotoxic activity). In contrast indirect activation was retained. We interpret these data to suggest that in the face of CyA immunosuppression CD4 + T cell effector function is ablated while CD8 + T cell function remains partially intact. The in vitro data suggest that the indirect pathway remains intact in this population of CyA resistant CD8 + T cells.

Impact of MHC Class II Incompatibility on Localization of Mononuclear Cell Infiltrates to the Bronchiolar Compartment of Orthotopic Lung Allografts

Chronic pathological changes in transplanted lungs are unique because they center on the airways. We examined the relative role of MHC class I and II antigens in causing bronchial pathology in orthotopic lung transplants to rats maintained on cyclosporin A (CsA). Transplants mismatched for MHC class II antigens had significantly more peri-bronchiolar infiltrates than MHC class I incompatible transplants. No significant increase in infiltrates was found in lung transplants incompatible for MHC class I plus II antigens compared to MHC class II antigens alone. Immunohistochemistry demonstrated that MHC class II antigen expression was confined to macrophages in MHC class I incompatible transplants, but was upregulated on bronchial epithelium in transplants with MHC class II incompatibilities. Vascular endothelium was notably devoid of MHC class II antigen expression in all transplants. However, both peri-bronchial and peri-vascular infiltrates were frequently cuffed by alveolar macrophages and type II pneumocytes that expressed MHC class II antigens. PCR analysis demonstrated that IFN-gamma and regulated on activation, normal T cells expressed and secreted (RANTES) were upregulated in MHC class II incompatible transplants. Thus, MHC class II incompatible orthotopic lung transplants in rats maintained on CsA immunosuppression undergo a bronchiolcentric upregulation of alloantigens.

Acute functional effects of cyclosporine-A and methylprednisolone treatment in adult rats exposed to transient ischemic stroke

The present study examined the neuroprotective effects of immunosuppressant cyclosporine-A (CsA) and anti-inflammatory methylprednisolone (MP) in a stroke model. Adult Sprague-Dawley rats were initially subjected to transient middle cerebral artery occlusion (MCAo) then randomly assigned to one of the following treatment conditions: low dose CsA, MP, low dose CsA plus MP, high dose CsA, or vehicle. Ischemic animals that received low dose CsA, MP or vehicle exhibited significant cognitive impairments, as revealed by passive avoidance and Morris water maze tasks, at days 1–3 after stroke. In contrast, ischemic animals that received high dose CsA exhibited near normal cognitive performance throughout the test period. Ischemic animals that received low dose CsA plus MP also showed significantly less cognitive deficits but such attenuation of stroke-induced behavioral impairments was only consistently reflected in the passive avoidance task, while performance in the Morris water maze task deteriorated over time. Histological analysis at 3 days post-stroke revealed that only those ischemic animals treated with high dose CsA had significantly reduced cerebral infarcts. These observations suggest that despite overt cerebral damage, alterations in simple, but not complex, cognitive tasks produced by MCAo could be ameliorated by low dose CsA when combined with MP.

Ciclosporina A e tacrolimus: uma revisão

INTRODUÇÃO: Monitorização terapêutica de imunossupressores ciclosporina A (CsA) e tacrolimus (FK506) é indispensável para manter níveis estáveis das drogas, evitando para o transplantado a perda do enxerto, no caso de baixas doses, ou a toxicidade, em altas doses, permitindo ajustes individuais. HISTÓRICO: Na década de 80, foi introduzida a utilização dos potentes imunossupressores CsA e FK506, revolucionando o transplante de órgãos com a diminuição da rejeição. MECANISMO DE AÇÃO: A CsA e o FK506 são inibidores da transcrição do primeiro sinal para ativação dos linfócitos T, apresentando estruturas químicas diferentes, mas mecanismos semelhantes. TOXICIDADE: Os principais efeitos relacionados à dose da CsA e do FK506 são a nefro e a neurotoxicidade. Estudos apontam o FK506 como droga alternativa à utilização da CsA porque aquele demonstrou menor nefrotoxicidade e uma reversibilidade dos efeitos neurotóxicos diante da redução da dose. METODOLOGIA ANALÍTICA: Na monitorização de rotina para a CsA, os imunoensaios, radioimunoensaio (RIA) e imunoensaio monoclonal com fluorescência polarizada (FPIAm) ocuparam o lugar da cromatografia líquida de alta eficiência (CLAE), detecção ultravioleta (UV). O método de referência para o FK506 é a CLAE com detecção por espectrometria de massa (LC/MS). Porém, metodologias como ensaio imunoenzimático de micropartículas (MEIA) ou enzyme linked imunosorbent assay (ELISA) têm sido utilizadas na rotina laboratorial. CONCLUSÃO: Parece existir uma tendência para a substituição terapêutica da CsA pelo FK506 no tratamento imunossupressor, porém ainda não se estabeleceu consenso nessa conduta. A metodologia analítica para a análise da CsA encontra-se bem estabelecida; para o FK506, estudos ainda são necessários.

Combined cyclosporine-A and methylprednisolone treatment exerts partial and transient neuroprotection against ischemic stroke

We investigated the neuroprotective effects of immunosuppressant cyclosporine-A (CsA) and the anti-inflammatory methylprednisolone (MP) in a stroke model. Adult Sprague–Dawley rats underwent middle cerebral artery (MCA) occlusion then were randomly treated with either: low dose CsA, MP, low dose CsA plus MP, high dose CsA, or vehicle. Ischemic animals that received low dose CsA, MP or vehicle displayed profound motor and neurological impairments at days 1–3 after stroke. In contrast, ischemic animals that received high dose CsA exhibited near normal motor and neurological functions throughout the test period. Of note, ischemic animals that received low dose CsA plus MP showed significantly less motor and neurological deficits at day 1, but thereafter displayed behavioral impairments. Histological analysis at 3 days post-stroke revealed that only those ischemic animals treated with high dose CsA had significantly reduced cerebral infarcts. This study is the first report to demonstrate partial and transient neuroprotection against stroke by low dose CsA when combined with MP.