FK506 Group Research Articles

Tacrolimus Toxicity Reverses the Inhibitory Effects of Renin Angiotensin System Blockade on PAI-1 Expression in Cardiac Tissue

Introduction: PAI-1 is a potent fibrosis promoting glycoprotein in a tissue dependent manner. We previously displayed that tacrolimus (FK506) toxicity increases vacuolar degeneration and arterial hyalinosis in cardiovascular tissue. FK506 toxicity induced transforming growth factor (TGF-β) expression. Renin angiotensin system (RAS) blockade partially reversed histopathological changes associated with FK506 toxicity. In the same model, we investigated the effects of FK506 and RAS blockade on PAI-1 expression. Materials and Methods: We examined cardiac expression of PAI-1 in a chronic FK506 toxicity model in Wistar rats. Study animals were divided into 4 groups. FK506 group was treated with FK506 intraperitoneally, FK506+Quinapril and FK506+Valsartan groups were treated Quinapril or Valsartan orally in addition to FK506. Control group was treated with saline. Immunohistochemical staining of cardiovascular tissue was semiquantitatively scored for PAI-1 expression. Results: FK506 significantly induced PAI-1 expression in the cardiovascular tissue compared to the control group (semiquantitative scores were 25±5 versus (vs) 49±21, p =0.01). Adding renin angiotensin system blockade with an angiotensin converting enzyme inhibitor (ACEI) or angiotensin receptor blocker (ARB) to FK506 increased FK506 induced PAI-1 expression. Semiquantitative PAI-1 expression scores were 49±21, 87±14 and 95±10 for FK506, FK506+ACEI, and FK506+ARB groups respectively (p

Calcineurin inhibitors and Clostridium difficile infection in adult lung transplant recipients: the effect of cyclosporine versus tacrolimus

BackgroundTacrolimus (FK506) has a superior immunosuppressive effect compared with cyclosporine (CSA) without a significant increase in generalized infectious complications. Differences in specific infections such as Clostridium difficile (CDI) have not been reported. We investigated the relationship between calcineurin inhibitors and CDI, hypothesizing that choice of calcineurin inhibitor (CSA or FK506) after lung transplantation would have no effect on the incidence of CDI. MethodsWe performed a retrospective chart review of lung transplant recipients between June 1, 2000, and December 31, 2005, at a single institution. Positive CDI assays through December 11, 2011, were also recorded. We used Student’s t- and chi-squared tests (α = 0.05) to compare CSA and FK506 groups. We calculated adjusted hazard ratios for CDI using Cox proportional hazard models. ResultsWe identified 217 lung transplant recipients: 106 patients in the CSA group and 111 patients in the FK506 group. A total of 31 patients (27.9%) in the FK506 group developed CDI postoperatively compared with 20 patients (18.9%) in the CSA group (P = 0.16). The adjusted hazard ratio for CDI in the FK506 group was not significantly higher (1.53; 95% confidence interval, 0.78–2.98). There was no significant difference in the intensive care unit or total length of stay, in-hospital incidence rate, time to first CDI episode, or recurrence rate between groups. ConclusionsThe CDI rates were not significantly higher in the FK506 group than the CSA group in our study. These data are consistent with previous studies on FK506 that show no increase in infectious complications over CSA, and demonstrate its continued safety in lung transplantation.

Use of FK506 and bone marrow mesenchymal stem cells for rat hind limb allografts.

Dark Agouti rat donor hind limbs were orthotopically transplanted into Lewis rat recipients to verify the effects of bone marrow mesenchymal stem cells on neural regeneration and functional recovery of allotransplanted limbs in the microenvironment of immunotolerance. bone marrow mesenchymal stem cells were intramuscularly (gluteus maximus) injected with FK506 (tacrolimus) daily, and were transplanted to the injured nerves. Results indicated that the allograft group not receiving therapy showed severe rejection, with transplanted limbs detaching at 10 days after transplantation with complete necrosis. The number of myelinated axons and Schwann cells in the FK506 and FK506 + bone marrow mesenchymal stem cells groups were significantly increased. We observed a lesser degree of gastrocnemius muscle degeneration, and increased polymorphic fibers along with other pathological changes in the FK506 + bone marrow mesenchymal stem cells group. The FK506 + bone marrow mesenchymal stem cells group showed significantly better recovery than the autograft and FK506 groups. The results demonstrated that FK506 improved the immune microenvironment. FK506 combined with bone marrow mesenchymal stem cells significantly promoted sciatic nerve regeneration, and improved sensory recovery and motor function in hind limb allotransplant.

Immunomodulatory effectiveness of tacrolimus in preventing epidural scar adhesion after laminectomy in rat model

There was no previous study about topical application of tacrolimus (FK506) could inhibit fibroblast proliferation and prevent epidural scar adhesion after laminectomy. We intended to illustrate the effect of FK506 on inhibiting fibroblast proliferation and preventing epidural scar adhesion after laminectomy in rat model. In our study, seventy-two rats were randomly divided into four groups (0.1mg/ml group, 0.05mg/ml group, 0.01mg/ml group and control group). Laminectomy was performed at Lumbar-1 level, and then different concentrations of FK506 and saline were applied to the laminectomy sites. Four weeks later the rats were killed and the epidural adhesion was evaluated. Macroscopic assessment, hydroxyproline content analysis, histological analysis and mRNA measurements were used to evaluate the effect of FK506 on reducing epidural scar adhesion. The results showed that FK506 could prevent epidural scar adhesion in a dose-dependent manner. Little epidural adhesions were seen in the laminectomy sites treated with 0.1mg/ml FK506. The hydroxyproline content, the number of fibroblasts, the mRNA expression level of IL-2 and TGF-β1 in 0.1mg/ml FK506 group were significantly less than those of 0.05mg/ml FK506 group, 0.01mg/ml FK506 group and control group. However, dense epidural adhesions were found in 0.01mg/ml FK506 group and control group. The hydroxyproline content and the number of fibroblasts in 0.01mg/ml group showed no significant difference compared with those of control group. In conclusion, topical application of 0.1mg/ml FK506 could inhibit fibroblast proliferation and prevent epidural scar adhesion after laminectomy in rat model.

Effects of neural stem cells transplantation and FK506 on pathologic and ultrastructural changes of injured spinal cord

Objective To investigate the impacts of neural stem cells (NSCs) transplantation on spinal pathology and ultrastructure after spinal cord injury (SCI) in rats and probe into the protective role of tacrolimus (FK506) on neural regeneration. Methods Compressive SCI at T8 was induced in the adult SD rats,which were randomly assigned to the control group,FK506 group,NSCs group and NSCs + FK506 group.The differences of neural regeneration in each group were compared at days 7,14,28 and 56 after injury by motor evoked potentials ( MEP),HE staining,immunohistochemical staining,ultrastructure observation and image analysis of the myelinated fiber. Results The MEP latency in the NSCs + FK506 group was significantly shorter than that in other groups at day 28 ( P ＜ 0.05 ).HE staining revealed that only local necrosis presented in the NSCs + FK506 group at day 56.More BrdU and NF-200 positive cells were detected with immunohistochemical staining in the other three groups as compared with the control group.Moreover,the positive cells in the NSCs + FK506 group also outnumbered the FK506 group and NSCs group.Electron microscope scan showed edema under the membrane of large myelin sheath in the control group,and classic new myelin sheath and neuraxis in the NSCs + FK506 group at day 56.The regeneration of the nerve fiber in the NSCs + FK506 group was better than that of other three groups (P ＜0.01 ). Conclusion After NSCs transplantation for SCI rats,the early combination use of FK506 can improve the pathology and ultrastructure of the regenerative nerve fiber and is conducive to nerve regeneration. Key words: Spinal cord injuries; Tacrolimus binding proteins; Stem cell transplantation; Pathology; Ultrastructure

Donor-derived transplant acceptance-inducing cells in composite tissue allotransplantation

Composite tissue allotransplantation (CTA) is a newly emerging field of transplantation. Immunological research in CTA has been intensified due to the recent clinical success of hand and face transplantation. Establishing immunological tolerance by adoptive transfer of exvivo cultured tolerance-inducing cell types is of growing interest. Transplant acceptance-inducing cells (TAICs) are a type of deactivated immunoregulatory macrophages. A total of 36 allogeneic hind limb transplantations in the rat were performed in six groups. Group A (Lewis (LW)→Brown-Norway (BN)) received Lewis-donor-derived TAICs locally (i.m.). Group B (LW→BN) received Lewis-donor-derived TAICs systemically (i.v.) and group C (Sprague Dawley (Sp-D)→BN) served as a control group receiving Lewis-donor-derived TAICs systemically (i.v.). Groups D (LW→BN), E (LW→BN), and F (BN→BN) also served as control groups with group D receiving no immunosuppression, group E receiving FK506 and prednisolone and group F receiving no immunosuppression with isograft transplantations (BN→BN). The timing of rejection was assessed by clinical observation and histological findings. Rejection of the allogeneic hind limb occurred on average 7.7 days after transplantation in group A and 7.4 days in group B. Rejection was significantly delayed (Log-rank test, p<0.01) compared to groups C and D, where rejection of the allogeneic hind limb occurred on average 5.8 days and 5.6 days after transplantation. No rejection was seen in groups E and F. For the first time, TAICs have been applied in a CTA model and demonstrated a significant immunosuppressive effect. Even though the immunomodulatory effect is relatively modest, the results of this study justify subsequent research on TAIC therapy to improve experimental and clinical outcome after CTA.

Mycophenolate mofetil and FK506 have different effects on kidney allograft fibrosis in rats that underwent chronic allograft nephropathy

BackgroundTacrolimus (FK506) is associated with renal fibrosis in long-term use. Mycophenolatemofetil (MMF) can also inhibit or attenuate the progression of renal fibrosis. This study aimed to determine the different effects of FK506 and MMF on fibrosis-associated genes in the kidney in rats that underwent chronic allograft nephropathy (CAN).MethodsFisher (F344) kidneys were orthotopically transplanted into Lewis rat recipients. All recipients were given Cyclosporin A (CsA) 10 mg/kg-1.d-1 × 10 day and were then randomly divided into three oral treatment groups (n = 9 in each group): (1) the vehicle group was given vehicle orally; (2) the FK506 group was given 0.15 mg/kg-1.d-1 FK506; and (3) the MMF group was given 20 mg/kg-1.d-1 MMF. At 4, 8, and 12 weeks post-transplantation, serum creatinine (SCr), collagen deposition, Connective tissue growth factor (CTGF), alpha smooth muscle actin (α-SMA) and E-cadherin expressions were determined and hematoxylin-eosin (HE) and Periodic acid-Schiff (PAS) stains were performed.ResultsRenal function progressively deteriorated and showed typical CAN morphology in the vehicle and FK506 groups, while SCr and inflammatory infiltration (Banff score) showed a significant decrease in the MMF group after 8 weeks post-transplantation compared with those in the other groups (p < 0.05). Furthermore, expression levels of CTGF and α-SMA in the MMF group were significantly reduced, and the down-regulated expression of E-cadherin was abated (p < 0.05).ConclusionsMMF showed favorable effects on renal interstitial fibrosis, thus efficiently retarding the progression of CAN.

Transfusion of Endothelial Cells with Lentivirus-Mediated Expression of Fas Ligand Prolonged Survival of Rat Liver Allograft

BackgroundImmunologically privileged sites have been shown to express Fas ligand (FasL) and may protect themselves by inducing apoptosis of infiltrating inflammatory cells. We asked whether the Fas/FasL interaction could be used to protect liver allograft from acute rejection. We proposed that endothelial cells that are resistant to Fas-mediated killing could be considered as a vehicle for expression of recombinant FasL. MethodsBased on the lenti-rFasl/puro expression system, constructs were designed that allowed endothelial cell-specific and continual expression of FasL. Endothelial cells with expression of FasL or viruses recombinant with FasL gene were transfused into portal vein of recipient rats during liver transplant surgery. Comparing groups of rats after liver transplant surgery using regular dose of FK506 and with no other treatment, we observed the aspartate aminotransferase and BIL value and survival of four groups of rat recipients. ResultsValues of AST and BIL in the cell and virus transfusion groups were between FK506 and contrast groups. The survival of cell and virus transfusion groups were longer than contrast group and shorter than FK506 group. ConclusionThis in vitro model shows that endothelial cells with expression of FasL or viruses recombinant with FasL gene transfusion can preserve liver function and prolong the survival time of liver allografts.

The Effect of Topical FK506 (Tacrolimus) in a Mouse Model of Allergic Rhinitis

The management of allergic rhinitis (AR) encompasses education, pharmacotherapy, immunotherapy, and surgery. FK506 (tacrolimus) is an immunosuppressant that inhibits allergic reactions. The purpose of this study was to reveal whether FK506 treatment reduces allergic inflammation in an AR mouse model and to elucidate the mechanisms. Forty mice were divided into four groups: control, AR, FK (FK506), and dexamethasone (DEX). All mice except for the control group were sensitized by an i.p. injection of ovalbumin (OVA). After sensitization, the FK and DEX groups were treated with FK506 and DEX intranasally. All sensitized mice were challenged intranasally with OVA. Allergic symptoms and tissue eosinophil counts were recorded. Interleukin (IL)-5, interferon gamma, and IL-10 levels in nasal lavage fluid (NALF) and serum OVA-specific IgE levels were measured. T-bet, GATA-3, and Foxp3 mRNA expression in splenic mononuclear cells were determined by real-time polymerase chain reaction. In the FK group and DEX group, allergic symptoms, serum OVA-specific IgE, tissue eosinophil counts, IL-5 in NALF, and GATA-3 mRNAs expression decreased (p < 0.05), and IL-10 in NALF and Foxp3 mRNAs expression increased compared with the AR group (p < 0.05). No significant difference was observed between the FK group and the DEX group. These results suggest that topical FK506 may reduce allergic inflammation and have potency equal to DEX in the AR model. This mechanism may involve not only Th2 cells but also regulatory T cells. Additional studies are needed on FK506, but in the future, we can consider FK506 as an alternative to topical steroids in the treatment of AR.

Effects of immunosuppressants, FK506 and cyclosporin A, on the osteogenic differentiation of rat mesenchymal stem cells

PurposeThe purpose of this study was to investigate the effects of the immunosuppressants FK506 and cyclosporin A (CsA) on the osteogenic differentiation of rat mesenchymal stem cells (MSCs).MethodsThe effect of FK506 and CsA on rat MSCs was assessed in vitro. The MTT assay was used to determine the deleterious effect of immunosuppressants on stem cell proliferation at 1, 3, and 7 days. Alkaline phosphatase (ALP) activity was analyzed on days 3, 7, and 14. Alizarin red S staining was done on day 21 to check mineralization nodule formation. Real-time polymerase chain reaction (RT-PCR) was also performed to detect the expressions of bone tissue-specific genes on days 1 and 7.ResultsCell proliferation was promoted more in the FK506 groups than the control or CsA groups on days 3 and 7. The FK506 groups showed increased ALP activity compared to the other groups during the experimental period. The ALP activity of the CsA groups did not differ from the control group in any of the assessments. Mineralization nodule formation was most prominent in the FK506 groups at 21 days. RT-PCR results of the FK506 groups showed that several bone-related genes-osteopontin, osteonectin, and type I collagen (Col-I)-were expressed more than the control in the beginning, but the intensity of expression decreased over time. Runx2 and Dlx5 gene expression were up-regulated on day 7. The effects of 50 nM CsA on osteonectin and Col-I were similar to those of the FK506 groups, but in the 500 nM CsA group, most of the genes were less expressed compared to the control.ConclusionsThese results suggest that FK506 enhances the osteoblastic differentiation of rat MSCs. Therefore, FK506 might have a beneficial effect on bone regeneration when immunosuppressants are needed in xenogenic or allogenic stem cell transplantation to treat bone defects.

Tacrolimus Induces Glomerular Injury via Endothelial Dysfunction Caused by Reactive Oxygen Species and Inflammatory Change

Background/Aims: The immunosuppressive drug tacrolimus (FK506) is used clinically to reduce the rejection rate in patients with kidney transplantation; however, the resultant nephrotoxicity remains a serious problem. In the present study we attempted to elucidate the mechanisms of glomerular injury induced by FK506 and the renoprotective effects of the angiotensin II receptor blocker telmisartan. Methods: Seven-week-old male Wistar rats were divided into three groups: vehicle group, FK506 group, and FK506 + telmisartan group. After 8 weeks, we assessed kidney function and renal morphological changes including oxidative stress. We also assessed the effect of FK506 in human glomerular endothelial cells (hGECs) with regard to reactive oxygen species (ROS). Results: FK506 induced ROS production via activation of NAD(P)H oxidase in the glomeruli. Expression of ICAM mRNA was increased in glomeruli from the FK506 group. These effects resulted in macrophage infiltration into the glomeruli. FK506 directly promoted NAD(P)H oxidase activity and accelerated production of ROS in hGECs. Conversely, cotreatment with telmisartan inhibited both NAD(P)H oxidase activity and production of ROS. Conclusion: These findings suggest that glomerular injury resulting from FK506 is caused by oxidative stress mediated by activation of NAD(P)H oxidase and that telmisartan exerts a renoprotective effect via antioxidative activity.

Micro-dose Ketamine on Optimized Patient-controlled Intravenous Analgesia in Advanced Cancer Patients

Objective Our study was to analyze the effect and safety of micro-dose ketamine plus fentanyl in patient-controlled intravenous analgesia(PCIA) for advanced cancer.Methods Eighty advanced cancer pain patients required were randomly divided into fentanyl group(group F)(n=20) and ketamine plus fentanyl groups(FK)(n=60).FK group included three groups of equal number,ie.FK1 Group(100 μg/ml fentanyl+ 1 mg/ml ketamine),FK2 Group(100 μg/ml fentanyl+ 2 mg/ml ketamine) and FK3 Group(100 μg/ml fentanyl+ 3 mg/ml ketamine).Analgesic consumption score,sedation score,respiratory depression,delirium and other complications were recorded during analgesia.Results In non-load condition,the onset time of analgesia in group FK2,FK3 was shorter than that in group F and group FK1.Fentanyl consumption in group F showed obviously increasing trend.Fentanyl consumption in group FK was constant during treatment and lower than that in group F.Group FK2 patients had better effect,almost did not need to regulate analgesia pump.Fentanyl consumption in FK3 was significant lower than that in other three groups,although with increased incidence of excessive sedation.No respiratory depression,hallucinations,delirium and other symptoms were observed.Conclusion Micro-dose ketamine could significantly reduce fentanyl consumption through protection of anti-opioid receptor,improve fentanyl tolerance,and enhance the quality of fentanyl PCIA analgesia in patients with advanced cancer.0.006 μg/kg.fentanyl plus(1.3 ±0.02) μg/kg ketamine was an ideal combination.

Ketamine as an adjunct to fentanyl improves postoperative analgesia and hastens discharge in children following tonsillectomy – a prospective, double‐blinded, randomized study

To evaluate the effect of ketamine, as an adjunct to fentanyl, on postoperative analgesia and duration of Postoperative Care Unit (PACU) stay, in children undergoing tonsillectomy. Ketamine, as an N-methyl-d-aspartate antagonist, has been recognized to have an opioid sparing effect. In addition, it does not depress respiration or affect airway tone. Hence, addition of ketamine could be potentially beneficial in children undergoing tonsillectomy, due to the high incidence of sleep apnea in these patients. In a double blinded, randomized trial, 60 ASA status I and II children between 2 and 7 years of age, scheduled to undergo elective tonsillectomy were recruited. They were randomly assigned to one of four groups to receive fentanyl 1 mcg·kg(-1) (F1 group), fentanyl 2 mcg·kg(-1) (F2 group), ketamine 0.5 mg·kg(-1) (K group), or fentanyl 1 mcg·kg(-1) plus ketamine 0.5 mg·kg(-1) (FK group) pre-incision. Postoperative pain was scored on arrival to the PACU and at 30, 60, and 90 min thereafter. Any incidence of nausea/vomiting and time to discharge from the PACU were also recorded. Important predictors found for postoperative pain on arrival to the recovery room are the group (P = 0.02) and duration of surgery (P = 0.02). Least square means and standard errors of pain scores on PACU arrival were 4.87±0.69, 3.04±0.68, 2.10±0.68 and 2.03±0.69 for F1, F2, K and FK groups, respectively. On group-wise comparison adjusted for surgical time, significant difference was detected between F1 and K (P = 0.02), and F1 and FK (P = 0.0048) groups. Marginal significance was detected in duration of PACU stay among groups (P = 0.08); F2 and FK group had a shorter PACU stay than F1 (P = 0.05 and 0.04 respectively). No significant difference was detected in the need for supplemental analgesia. We conclude that the administration of ketamine 0.5 mg·kg(-1) with 1 mcg·kg(-1) fentanyl in children undergoing tonsillectomy may improve postoperative pain control without delaying home discharge.

Effects of a calcineurin inhibitor, FK506, and a CCR5/CXCR3 antagonist, TAK-779, in a rat small intestinal transplantation model

The effects of FK506, and TAK-779, antagonists of CCR5 and CXCR3, were investigated using a rat intestinal transplantation model. Small intestines from DA rats were heterotopically transplanted into LEW rats. The recipients were treated with FK506 (1 mg/kg/day, day 0–5) and TAK-779 (10 mg/kg/day, day 0–10). Graft survival and immunological responses to these materials were estimated by mixed lymphocyte reactions and IFN-γ production. The expression of chemokine receptors on lymphocytes was also examined. The average duration of survival was 7.0 ± 0.3, 12.0 ± 1.0, 9.8 ± 0.5 and 18.0 ± 1.5 days in the allogeneic, FK506, TAK-779 and the two-drug combined groups, respectively. Cell proliferative responses and IFN-γ production were suppressed to a significant extent in the FK506 group compared with the TAK-779 group. In addition, the two-drug combination showed a tendency for stronger suppression than FK506 alone, correlated with in vivo and histopathological data. The numbers of both CD4 + and CD8 + cells were significantly suppressed in the blood of the recipients of both the FK506 and the TAK-779 groups, and in Peyer's patches of the graft of the TAK-779 group, but the FK506 group was not, as evidenced by FACS analysis. In addition, double-staining of graft-infiltrating lymphocytes showed a significant reduction in lymphocyte numbers, expressing CCR5 and CXCR3 in the TAK-779 group, but not evident in the FK506 group, compared to the allogeneic group. While FK506 suppresses cell proliferation and effecter function, it has less effect on the expression of CCR5 and CXCR3 in lymphocytes. Further exploration of the effects of a combined therapy with TAK-779 could represent a novel treatment for intestinal transplantation.

The acute effects of FK-506 on renal haemodynamics, water and sodium excretion and plasma levels of angiotensin II, aldosterone, atrial natriuretic peptide and vasopressin in pigs.

FK-506 has been shown to be an effective immunosuppressive drug with possible nephrotoxic side effects. In this study we have investigated the acute effects of FK-506 on renal haemodynamics, water, sodium and lithium excretion rates and plasma levels of angiotensin II, aldosterone, atrial natriuretic peptide (ANP) and vasopressin in 29 anaesthetized Lancaster/Yorkshire female pigs. A continuous intravenous infusion was given over a 2-h period to 4 groups: A: 0.075 mg kg-1 (n = 7), B: 0.15 mg kg-1 (n = 8), C: 0.3 mg kg-1 (n = 6) and P: placebo vehicle (n = 8). Glomerular filtration rate (GFR) and renal plasma flow (RPF) were measured by the constant infusion clearance technique using 125-I-iothalamate and 131-I-hippuran as reference substances. Hormonal parameters were measured by radioimmunoassay. In all three FK-506 groups, fractional lithium excretion was significantly decreased 2 h after FK506 infusion (P: +0.4%, A: -8.8% (P < 0.05), B: -12.9% and C: -11.2% (P < 0.01 for both). Mean arterial blood pressure (MBP) was significantly increased in the two highest dosage groups (B,C) at 2 h of infusion: (MBP; P: +2.9%, A: +3.5%, B: +12.0%, C: +15.3% (P < 0.01 for both). GFR and RPF showed minor and inconsistent changes while all other parameters measured showed similar or no changes. In conclusion, acute infusion of FK-506 to pigs does not change overall renal function significantly, but increases mean arterial blood pressure and decreases fractional excretion of lithium.

Return of lymphocyte immunoreactivity after discontinuation of a brief course of Prograf, Cyclosporine, or Sirolimus in normal subjects. (169.13)

Abstract The mainstay of immunosuppression is Prograf (FK), Cyclosporine (CSA), and Sirolimus (SRL). These drugs target T cells which are pivotal in transplant rejection. Little is known about their effects on length of T cell suppression once stopped. We hypothesized that T cell reactivity would return later with SRL compared to CSA or FK. 18 health volunteers were recruited. 8 received FK, 3 received CSA, and 7 received SRL. FK and CSA were taken twice daily for 3.5 days and SRL once daily for 4 days. Drugs were titrated to respective troughs (FK 8-10 mg/L, CSA 150-200 mcg/L, SRL 10-15 ng/ml). Blood were drawn at 0, 12, 24, &amp; 48 hrs after last dose. CD4 cells were isolated using CD4 isolation beads, stimulated with CD3/28 beads, and cultured in 96 well plates. Cell proliferation and ATP concentration were measured using BrdU labeling and Cylex assay respectively. At 24 hrs, ATP concentration fold change between FK and SRL by Cylex was not statistically significant (1.0 vs. 0.8, p=0.2). T cell proliferation measured by BrdU compared to time 0 was significant in 2/8 patients at 12 &amp; 24 hrs and 4/8 patients at 48 hrs in FK group, 1/3 patients at 12 &amp; 48 hrs in CSA group, and 6/7 patients at 12 &amp; 48 hrs in the SRL group (p&amp;lt;0.05). Proportions were not significant but did trend towards significance in SRL compared to FK at 12 hrs (p=0.07). In conclusion, SRL was not associated with longer immunosuppression of T cells stimulated in-vitro compared to patients taking FK and CSA.

FK506 promotes repair of injured spinal cord pathway after neural stem cell transplantation

Objective To observe the effect of tacrolimus(FK506)in promoting repair of the injured spinal cord pathway after neural stem cell transplantation in rats. Methods A neurysm clip was used to compress the T8 spinal cord segment of SD rats under microscope to establish model of spinal cord injury.The rats were randomly divided into three groups seven days after injury,ie,control group (injection of normal saline at the injury center),transplantation group(injection of neural stem cells,NSCs,at the injury center),FK506 group(injection of NSCs at the injury center plus 7 days of intrapernerve conduction was compared by using the Basso-Beatfle-Bresnahan (BBB) scale,BDA tracing,somatosensory evoked potential(SEP)and motor evoked potentials(MEP)monitoring at 1,2,4 and 8weeks. Results The motor function of the hind limb after injury was recovered in various degrees with time,with the most significant recovery at 4 weeks.The BBB score reached 6,the maximum at 8 weeks.BDA tracing showed that some nerve fibers were found crossing the injured center of the spinal cord one week later in FK506 group and cell transplantation group,that BDA-positive labeled corticospinal tract fibets were seen across the injury site in all groups by the end ofthe eight weeks.In the FKS06 group,the regeneration could be observed even as 1.7 cm away from tlle injury center.SEP latency was significantly shorter in the FK506 group after two weeks(P<0.05)and the MEP latency in the FK506 group was shortened significantly at four weeks compared with the other groups(P<0.05),indicating that the immunosuppressants could promote the recovery of the injured spihal cord,shorten the latency of SEP and MEP，improve SEP at early stage and MEP at late stage．Conclusions Systemic application immunosuppressive agents FK506 plays an important role in neuroprotection and neurotrophy，which promotes the repair of the injured spinal cord after neural stem cell transplantation． Key words: Spinal cord injuries; FK506; Stem cell transplantation; Evoked potentials

Lifetime behavioural changes after exposure to anaesthetics in infant rats

The aim of this study was to assess the effect of acute use of general anaesthetic with or without a surgical procedure, at post-natal day 14 (P14), on behavioural responses in the short-, medium- and long-term, evaluated in open field (OF) and elevated plus-maze (EPM) tests. Fourteen-day-old male Wistar rats were divided into two experimental designs (ED): inhalation and intravenous anaesthetic, and these groups were subdivided into: 1st ED – control (C), isoflurane (ISO), isoflurane/surgery (ISO-SUR); 2nd ED – control (C), fentanyl/S(+)-ketamine (FK) and fentanyl+ketamine-s/surgery (FK-SUR). In the OF the following were found: (a) in the 1st ED: an increase in the locomotor activity in the ISO group at P14, and ISO and ISO-SUR groups at P30; the ISO-SUR group showed a reduced latency to leave the first quadrant at P30 and P60; (b) in the 2nd ED: FK and FK-SUR groups presented increased locomotor activity at P30, and the FK group showed a reduction in the number of faecal boluses. In the EPM the following were found: FK and FK-SUR groups presented an increase in the number of non-protected head-dipping (NPHD) movements and in the number of entries and time spent in open arms at P30; the FK group showed an increased number of protected head-dipping movements, NPHD and entries and time spent in the open arms at P60. The behavioural changes observed may be related to locomotor activity (1st ED) and anxiety level (2nd ED) and they may result from changes in neurotransmitters/hormones (DA, 5HT, CRH) and glutamate/NMDA receptors, respectively.

951 Tacrolimus and Leu-Ile Inhibit Hypoxic-Ischemic Encephalopathy in Newborn Rats

We used a neonatal rat hypoxia ischemia (HI)/ reperfusion brain damage model and investigated the neuroprotective effects of the immunosuppressant tacrolimus (FK506) and the hydrophobic peptide Leu-Ile, which has an active site similar to that of FK506 but is not an immunosuppressant. Method: HI was induced in 7- or 8-day-old Wister rat pups by transient right carotid artery occlusion, followed by exposure to 8% O. After HI insult, the rats were intraperitoneally administered FK506, Leu-Ile, a combination of the 2 drugs, or vehicle. Twenty-four hours or 7 d after HI, we analyzed the decreasing ratio of cerebral hemispheric weights and then histologically examined the brain tissue by HE, Iba-1, and caspase-3 staining. Body weight gain was also monitored. Result: The decreasing ratio of cerebral hemispheric weights in the FK506, Leu-Ile, and combination groups was lower than that in the vehicle group, but this ratio was significantly different only between the vehicle and FK506 groups. Pathological analysis showed that neurocytic injury had a strong suppressive effect in the FK506 and combination groups and a mild effect in the Leu-Ile group. During the 7-d experiment, the FK506 group showed lower body weight gain than the other groups. Conclusion: Combination treatment with Leu-Ile and FK506 had a stronger neuroprotective effect and lesser side effects; thus, we concluded that combination therapy might have potential for clinical application in neonatal HIE.

Antigen Sampling on the Peyer's Patches in a Murine Small Bowel Transplantation Model

Aim This study investigated changes in the mucosal barrier of transplanted intestines with particular emphasis on antigen sampling by Peyer's patches (PPs). Methods Heterotopic small bowel transplantation (SBTx) was performed as described previously. C57BL/6 mice were used as donors and BALB/c (allogeneic) or C57BL/6 mice (syngeneic) as recipients. Tacrolimus (FK506) or saline control was administered to the recipients for 2 weeks. Four groups included in this study were: syngeneic with or without immunosuppression (SYN and SYN + FK506, respectively) and allogeneic with or without immunosuppression (ALLO and ALLO + FK506, respectively). Animals were sacrificed weekly after SBTx to evaluate microfold (M) cells within PPs and for routine histology. By the third postoperative week, recipients were subjected to an intestine loop model to examine the uptake of microbeads by M cells as well as expression of Toll-like receptor 2 (TLR2) protein in the PPs with or without a TLR2 agonist challenge. We also measured occludin expression on follicle-associated epithelium (FAE) of PPs in the grafts. Results Transportation of microbeads through the PPs of the grafts increased in the ALLO + FK506 group compared with that in the SYN or SYN + FK506 group. This finding was accompanied by increased expression of TLR2 in the PPs and a gradually increased number of M cells following SBTx. However, occludin expression patterns on the FAE of the PPs in the grafts were similar among SYN, SYN + FK506, and ALLO + FK506 groups. Nevertheless, as transportation of microbeads and TLR2 expression in the PPs of the grafts was enhanced once exposed to Pam3Cys-SKKKK, similar results were not seen in the ALLO + FK506 group. Conclusions Our study revealed that the mucosal barrier of intestinal grafts is altered under alloreactivity as evidenced by enhanced antigen sampling. Such a change may provide a pathway for translocation of microorganisms in the lumen.