FK506-treated Group Research Articles

Induction of liver transplant immune tolerance in an outbred rat strain model using tacrolimus

BackgroundOrthotopic liver transplantation is the only option for patients with end-stage liver disease and hepatocellular carcinoma. Post-transplant immunosuppressive therapy is important to prevent graft failure. We investigated the effectiveness of tacrolimus (FK506) and their mechanisms for liver transplant immune tolerance in an outbred rat LT model.ResultsTo investigate the therapeutic effect of the FK506 on outbred rat LT model, FK506 and postoperative therapy were administered subcutaneously once or twice daily to transplanted rats. Histopathological and immunohistochemical analyses were conducted for all groups. The regulation of inflammatory cytokine signaling in the spleen was analyzed by flow cytometry. FK506 attenuated allograft rejection and increased survival in rat orthotopic liver transplantation models. The FK506-treated group had reduced serum levels of alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase. Furthermore, FK506 decreased the expression of inflammatory cytokines and the activation of pathogenic Th1 and Th17 cells in the liver.ConclusionsTaken together, we revealed that FK506 ameliorated strong allograft rejection in outbred liver transplantation model by anti-inflammatory effect and inhibitory peroperty of pathogenic T cells.

Tacrolimus pretreatment on liver ischemia reperfusion injury in rats

Objective To determine the effects of tacrolimus (FK506) pretreatment on the liver ischemia reperfusion (IR) injury. Methods 32 mature SD rats were randomly assigned into four groups, which were sham-operated group (S), ischemia reperfusion group (IR), low-dose FK506-treated group (L) and high-dose FK506-treated group (H). After the treatment of liver ischemia for 60 minutes and reperfusion for 6 hours, the levels of serum ALT and AST in rats were tested. The TNF-α and IL-1β levels were evaluated by enzyme linked immunosorbent assay. Liver damage was assessed by paraffin sections stained with H&E. The quantitative real-time PCR, the immunohistochemistry and Western blot were used to detect the expression of HMGB1 mRNA and protein with or without FK506 pretreatment. Results The levels of serum ALT [(424.0±137.4)U/L, (291.0±42.0)U/L], AST [(554.2±127.7)U/L, (410.2±7.0)U/L], TNF-α [(115.1±49.0) ng/L, 120.4±28.5) ng/L] and IL-1β [(424.5±105.2) ng/L, (612.1±49.6) ng/L] decreased markedly in the group L and group H compared with the group IR (P 0.05). Conclusion FK506 pretreatment can protect the liver by reducing the expression of HMGB1, inhibiting the release of inflammatory cytokines and alleviating cell necrosis after the liver ischemia reperfusion injury in rats. Key words: Tacrolimus; Liver; Ischemia reperfusion; High mobility group box 1 protein, HMGB1; Liver protection

Influence of Tacrolimus (FK506) on Nerve Regeneration Using Allografts: A Rat Sciatic Nerve Model

FK506 is an immunosuppressant agent used to prevent rejection after organ transplantation. The aim of the present study was to assess effects of tacrolimus (FK506) on peripheral nerve regeneration using allografts in a rat sciatic nerve model. Thirty male white Wistar rats were divided randomly into a normal control (NC) group (n = 10), an allograft (ALLO) group (n = 10), and an FK506-treated (ALLO/FK506) group (n = 10). In the NC group, the left sciatic nerve was exposed through a gluteal muscle incision and, after homeostasis, the muscle was sutured. In the ALLO group, the left sciatic nerve was exposed through a gluteal muscle incision and transected proximal to the tibioperoneal bifurcation, where a 10-mm segment was excised. The same procedure was performed in the ALLO/FK506 group. The harvested nerves of the ALLO group served as allografts for the ALLO/FK506 group and vice versa. The NC and ALLO groups received sterile olive oil 300 μL intraperitoneally once a day for 1 week and the ALLO/FK506 group received FK506 300 μL (1 mg/kg) intraperitoneally once a day for 1 week. Behavioral, functional, and biomechanical recovery and gastrocnemius muscle mass showed earlier regeneration of axons in the ALLO/FK506 than in the ALLO group (P < .05). Histomorphometric and immunohistochemical studies also showed earlier regeneration of axons in the ALLO/FK506 than in the ALLO group (P < .05). Administration of FK506 could accelerate functional recovery of the sciatic nerve after nerve allografting. It could have clinical implications for the surgical management of patients after facial nerve transection.

Tacrolimus (FK506) suppresses TREM-1 expression at an early but not at a late stage in a murine model of fungal keratitis.

PurposeTo investigate the efficacy and mechanism of tacrolimus(FK506), which is a novel macrolide immunosuppressant, in inhibiting triggering receptor expressed on myeloid cells-1 (TREM-1) expression in a murine keratitis model induced by Aspergillus fumigatus. MethodTREM-1 was detected in 11 fungus-infected human corneas by quantitative real-time PCR (qRT-PCR). RAW264.7 macrophages were divided into four groups, which received treatment with zymosan (100 µg/ml), zymosan (100 µg/ml) + mTREM-1/Fc protein (1 µg/ml), or zymosan (100 µg/ml) + FK506 (20 µM) or negative-control treatment. After this treatment, the expression of TREM-1, interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα) was assayed using qRT-PCR and ELISA. The mouse model of fungal keratitis was created by intrastromal injection with Aspergillus fumigatus, and the mice were divided into 2 groups: group A received vehicle eye drops 4 times each day, and group B received 4 doses of FK506 eye drops each day. Corneal damage was evaluated by clinical scoring and histologic examination,and myeloperoxidase (MPO) protein levels were also detected by ELISA. The expression of TREM-1, IL-1β and TNFα was then determined at different time points using qRT-PCR and ELISA.ResultsTREM-1 expression dramatically increased in the human corneas with fungal keratitis. In contrast, FK506 reduced the expression of TREM-1, IL-1β and TNFα in RAW264.7 macrophages stimulated with zymosan. In the mouse model, at day 1 post-infection, the corneal score of the FK506-treated group was lower than that of the control, and polymorphonuclear neutrophil (PMN) infiltration was diminished. TREM-1, IL-1β and TNFα expression was significantly reduced at the same time point. However, the statistically significant differences in cytokine expression, clinical scores and infiltration disappeared at 5 days post-infection.ConclusionsFK506 may inhibit the inflammation induced by fungi and alleviate the severity of corneal damage at an early stage of fungal keratitis by downregulating TREM-1 expression.

Effects of FK506 on long-term potentiation observed by optical imaging in organotypic hippocampal slice culture

Electrophysiological and biochemical assays were used to observe long-term changes in synaptic efficacy following electrical and/or pharmacological manipulation of synaptic function. We investigated the effects of FK506 on synaptic plasticity in organotypic hippocampal slice culture (OHSC) using optical imaging system. Hippocampal slices were prepared from 6-7 day-old rats using a tissue chopper and placed on a membrane insert. 18 hr after kainic acid (KA) treatment, significant delayed neuronal death which was quantified by propidium iodide, NeuN, and TUNEL staining was detected in CA3 area at 24 hr after KA treatment. The neuronal death was prevented significantly at 24 hr after 0.1 μM FK506 treatment. In the electrophysiology study, optical signals were observed by long-term potentiation (LTP) followed by the stimulation of the Schaffer collateral pathway. The improvement in amount of LTP was found in the FK506-treated group. These results suggest that FK506 may have a beneficial role in recovery of synaptic efficacy following KA-induced neuronal injury.

The Immunosuppressive Agent FK506 Prevents Subperineurial Degeneration and Demyelination on Ultrastructural and Functional Analysis

Several kinds of injury models, such as crush, transection and graft repair have been well studied in terms of neuroprotective effect of FK506. However, definitive experimental studies are lacking on focal degeneration or ischemia. In the present study, our goal was to investigate the effect of FK506 on functional recovery of the sciatic nerve after focal ischemia, produced by stripping of the epineurial vessels. A total number of 48 Wistar rats were used for this purpose and divided into four groups (control, sham-operated, FK506-treated, and Vehicle-treated). Sciatic nerves were approached by femoral and gluteal muscle splitting. Then, epineurial vessels around the sciatic nerve were stripped in the FK506-treated and Vehicle-treated groups. After operation, 5mg/kg/day FK506 administration was initiated by subcutaneous injection until animal sacrifice. The same volume of saline was administrated to the vehicle-treated group. The functional and sensory recoveries were tested by walking pattern analysis and pinch test in every postoperative week. The animals were sacrificed in the end of the fourth postoperative week and sciatic nerve samples were harvested and processed for electron microscopic evaluation. Our data revealed that FK506 administration showed beneficial effect on subperineurial degeneration/demyelinization from functional, sensorial, and ultrastructural points of view. The sciatic nerve samples in the FK506-treated group had several remyelinated fibers compared to the vehicle-treated group. Our literature searches revealed that FK506 administration has not, to our knowledge, been studied in focal ischemic degeneration produced by stripping of the epineurial vessels.

Effect of intrathecal FK506 administration on intraorbital optic nerve crush: an ultrastructural study

Objective: Trauma to the optic nerve caused by fractures of the midface and (or) skull base has been simulated by an optic nerve crush injury model. Because the intraorbital segment of the optic nerve is surrounded by subarachnoidal cerebrospinal fluid and dura mater, we aimed to study the influence of intrathecal tacrolimus (FK506) administration after optic nerve crush injury and to determine its role in optic nerve protection or sparing after injury. Study Design: Experimental study. Participants: All optic nerves of the animals were included in the study. Methods: A total of 48 female Wistar rats were randomly divided into 4 groups (control, sham operated, FK506 treated, and vehicle treated). In vehicle- and FK506-treated groups, intrathecal catheter implantation and crush injury to the intraorbital part of the optic nerve were performed and then the animals were treated intrathecally. The optic nerve samples were harvested on the 30th postoperative day. Optic nerve appearances were analyzed qualitatively. Results: Light and electron microscopic evaluations revealed that numerous damaged myelin residues were present in the vehicle-treated group, whereas fibres of the optic nerve showed a well-shaped appearance in the FK506-treated group. Conclusion: We propose that such an intrathecal administration route and small-dose regimen should be used to obtain lesser immunosuppression and neurotoxicity and higher protection or sparing after injury.

Effect of FK506 administration after obturator nerve injury: a functional and ultrastructural study.

The frequency of obturator nerve damage due to pelvic diseases, fractures or gynecologic procedures is uncertain. In the present study, we investigated the effect of FK506, a potent macrolide antibiotic and immunosuppresant, on obturator nerve recovery at morphological and functional levels. Forty female Wistar rats were randomly divided into four groups (control, sham, FK506-treated, vehicle-treated). In half of animals (FK506-treated and vehicle-treated) an obturator nerve crush (30 seconds clamp) was created. In FK506-treated group FK506 administration (1 mg/kg/day, subcutaneously) was performed on each postoperative day. All the rats were functionally evaluated by pinch and adduction tests preoperatively and postoperatively at one, two, three and four weeks after nerve injury. On the 28th postoperative day obturator nerve samples were collected and analyzed qualitatively by light and electron microscopy. FK506 treatment resulted in dramatic improvement in nerve function and in the ultrastructure of nerve fibers suggesting its therapeutic potential in traumatic obturator nerve injury.

Effects of Intrathecal Administration of FK506 after Sciatic Nerve Crush Injury

Although various administration routes of FK506 have been published, intrathecal administration of FK506 has not previously been reported in the literature. A daily dose of 0.05 mg/kg of FK506 was given (a small dose compared with those reported in the available literature). The authors used this small dose to obtain lower immunosuppression and neurotoxicity, and a higher axonal regeneration rate. A total number of 40 female Wistar rats were used and randomly divided into four groups: control, sham, FK506-treated, and vehicle-treated. Sciatic nerve regeneration was evaluated by walking track analysis, an electrostimulation test, and light microscopic evaluation. There was a statistically significant difference ( P < 0.05) between FK506-treated and vehicle-treated groups at the end of 6 weeks according to both the walking track analysis and the electrostimulation test. Comparing the stimulus thresholds of the sham and FK506-treated group, no significant difference ( P > 0.05) was observed. Evaluation of the data revealed that FK506 had a beneficial effect on sciatic nerve regeneration.

The role of cyclophosphamide and granulocyte colony‐stimulation factor in achieving high‐level chimerism in allotransplanted limbs

The establishment of a high-level of chimerism may be the most stable strategy for donor-specific tolerance. The purpose of this study was to evaluate the efficacy of a new protocol using cyclophosphamide (CYP) and granulocyte colony-stimulation factor (G-CSF) to induce high-level chimerism following rat whole-limb allotransplantation. Seventy-three whole-limb allotransplants from LacZ transgenic rats to LEW rats were performed. CYP was injected at day 2, and G-CSF was given from day 0 to 3. Nontreated limb allografts were rejected after 4.2 days. In FK506-treated group for 28 days, the survival time was prolonged to 64 days. In the group treated with CYP/G-CSF, limb allografts were rejected after 5.4 days and 5 of 15 recipients showed acute lethal graft-versus-host disease (GVHD). Polymerase chain reaction (PCR) study showed a high level of chimerism even within 1 week after transplantation. Fourteen of 30 recipients given CYP/G-CSF/FK506 died within 2 weeks. The limb survival was significantly prolonged, however, with three grafts surviving more than 300 days. Seven recipients (24%) showed chronic GVHD. A high-level of chimerism was maintained when limb allografts were not rejected by recipients. Limb allografting could function as a vascularized carrier for bone marrow transplantation, provide a continuous source of donor cells and contribute to a high level of chimerism in the recipient. Pretransplant CYP followed by G-CSF and FK506 treatment significantly prolonged the survival of limb allografts but frequently caused chronic GVHD in the recipients.

Immunosuppression with FK506 has no influence on fracture healing in the rat

Immunosuppressant drugs like cyclosporine A and FK506 are widely used for solid organ transplantation. They are accelerating bone remodeling but cause net bone loss. The aim of this study was to investigate the effect of FK506 on fracture healing in the rat. Eighty Lewis rats were divided into four groups, which received FK506 (1 mg/kg BW) or no treatment for 2 or 4 weeks, beginning after production of a closed, nondisplaced unilateral tibial fracture. Radiographic, histological, and biomechanical studies were used to evaluate fracture healing and histomorphometric analysis of the tibial metaphysis of the intact contralateral side was performed. Radiographs revealed no difference of the healing of the control fractures compared with the fractures in the FK506-treated group at 2 and 4 weeks. The mechanical parameters of the tested contralateral intact tibiae and of the fracture callus demonstrated no difference between control and immunosuppressed animals. Tibial bone histomorphometry revealed increased measures of bone formation and bone resorption, accompanied by a significant reduction of percent trabecular area. At 4 weeks, the fractures showed osseous healing with woven bone at the fracture site and only minimal amounts of cartilage. Histological grading was not different between the control and the FK506 group at both time points. We conclude that systemic application of FK506 has no biomechanical and histological effects of experimental fracture healing in the rat. However, resorption far in excess of formation leads to a net bone loss in the trabecular bone of the tibia that has no effect on the stability of the intact bone.

Regulation of bone metabolism in immunosuppressant (FK506)-treated rats

After organ transplantation, severe osteoporosis is occasionally seen, and the use of immunosuppressants is thought to be one of the causes of such osteoporosis. In the present study, we investigated the effects of FK506 monotherapy on bones and determined the mechanism of onset of osteoporosis, both by assessing chronological changes in bone metabolism and by identifying factors that facilitate bone resorption. In 8-week-old male Sprague-Dawley rats, FK506 (1 mg/kg) was injected intraperitoneally every day for 5 weeks (FK506-treated group), and for comparison, physiological saline was administered in the same manner in a control group of rats. Serum and urine samples were collected at weeks 0, 1, 3, and 5 of administration. The femur and tibia were collected within 24 h of the final administration. When compared to the control group, findings on three-dimensional micro-computed tomography of the femur for the FK506-treated group showed a significant decrease in trabecular bone volume. The level of serum osteocalcin in the FK506-treated group at week 1 of administration was significantly higher than the control. Throughout the administration period, the sum of urinary pyridinoline (PYD) and deoxypyridinoline (Dpd) was significantly higher in the FK506-treated group. Of the various bone resorption factors tested, the level of serum parathyroid hormone (PTH) in the FK506-treated group was significantly higher than the control at week 3 of administration. The results of the present study confirmed that FK506 monotherapy in rats induced high-turnover osteoporosis. Soon after the start of FK506 administration, bone formation and resorption were elevated, and PTH appeared to have been involved in the maintenance of the elevated bone resorption.

Mild hypothermia enhances the neuroprotective effects of FK506 and expands its therapeutic window following transient focal ischemia in rats

FK506 (tacrolimus), an immunosuppressant, reportedly reduces ischemic brain injury following transient middle cerebral artery occlusion (MCAO) in rats. The authors previously reported that the therapeutic window of FK506 in this model is more than 1 h, but less than 2 h. The aim of the present study is to determine whether mild hypothermia (35 °C) enhances the neuroprotective effects of FK506 and expands its therapeutic window. Sprague–Dawley rats were subjected to 2 h MCAO followed by 24 h reperfusion. Animals were randomly divided into four groups: (I) vehicle-treated normothermic group; (II) FK506-treated normothermic group; (III) vehicle-treated hypothermic group; (IV) FK506-treated hypothermic group. Animals received a single injection of FK506 (0.3 mg/kg) or vehicle intravenously at 2 h after ischemic induction. During ischemia, temporal muscle and rectal temperatures were maintained at 37 °C in the normothermic animals and at 35 °C in the hypothermic animals. Infarct volumes and neurological performance were evaluated at 24 h after reperfusion. The combination of FK506 and mild hypothermia significantly reduced infarct volume (cortex, −61%; striatum, −31%) and edema volume (cortex, −57%; striatum, −41%), while mild hypothermia or FK506 alone failed to improve ischemic brain damage. Furthermore, this combination also provided for the best functional outcome. These results demonstrate that the combination of FK506 and mild hypothermia significantly reduces ischemic brain damage following transient MCAO in rats, and expands the therapeutic window for FK506. This therapy may be a new approach for treatment of acute stroke.

Bimodal dose-dependence of FK506 on the rate of axonal regeneration in mouse peripheral nerve.

FK506 has been shown to enhance the rate of axonal regeneration after peripheral nerve lesions. However, quite variable doses of FK506 have been used in different animal studies. We examined the dose-dependence of FK506 on the rate of axonal regeneration after crush lesion of the mouse sciatic nerve. Mice received daily subcutaneous injections of FK506 at 0.2, 0.5, 1, 2, 5, or 10 mg/kg for 7 days after lesioning. A control group was injected with saline. The distance that regenerative axons advanced from the crush site was measured by the pinch test at 2, 4, and 7 days. Regenerating axons reached greater mean distances in all FK506-treated groups compared to the control group. The fastest regeneration rate was found at 5 mg/kg (12% increase over controls), although the 0.2 and 2 mg/kg doses achieved similar regeneration rates. In contrast, intermediate doses (0.5 and 1 mg/kg) and a higher dose (10 mg/kg) were not different from controls. Calcitonin gene-related peptide immunohistochemical labeling of regenerating axons yielded similar results to those found with the pinch test. Based on our finding of a double peak in the dose-response for FK506, it is hypothesized that at least two mechanisms of action (perhaps corresponding to distinct functional binding sites) are evoked at different concentrations of the drug to accelerate nerve regeneration. These results have clinical implications for the pharmacological treatment of nerve injuries while avoiding immunosuppressive effects and for the design of related drugs with more specific activities.

FK506 suppressed the inflammatory change of EAM in SJL/J mice

Experimental autoimmune myositis (EAM) is a good model of human inflammatory myopathy. We induced EAM in SJL/J mice by injection with myosin and treated inflammatory changes with FK506. The mice developed inflammatory changes after the fifth myosin injection. After treatment with FK506, inflammation was suppressed and central nuclei of the muscle fibers increased. These findings indicate that FK506 is effective in the treatment of EAM. The data suggests that FK506 inhibits interaction with calcineurin. Intercellular adhesion molecule-1 (ICAM-1) positive cells were present in the inflammatory and non-inflammatory areas of EAM. The FK506-treated group stained more weakly for ICAM-1 than the untreated EAM group.

Role of CD4+ and CD8+ T cells in early and late acute rejection of small bowel allograft

Background/Purpose: Results of small bowel transplantation remain unsatisfactory because of severe immune rejection. The current study aims to elucidate the role of activation of CD4+ and CD8+ T cells in early and late acute rejection of small bowel allograft and, hence, provide the immunologic basis for developing new therapeutic strategies. Methods: We used an MHC fully mismatched (DA to Lewis) heterotopic rat small bowel transplant model and a unique FK506-based immunosuppressive regimen, which suppresses early acute rejection but does not prevent late acute rejection. Flow cytometric analysis was used to quantitate the number of activated CD4+ and CD8+ T cells in graft and host mesenteric lymph nodes. Results: The survival (mean ± SD) of intestinal allograft was significantly prolonged, from 6.6 ± 0.84 days for the untreated group to 40.7 ± 14.1 days for the FK506-treated group. Activation of CD4+ cells was suppressed significantly in the FK506-treated group on postoperative day 7 compared with the untreated group (29.4% ± 3.55% v 52.83% ± 11.9%; P <.01). Activation of CD8+ cells was similarly suppressed (31.5 ± 10.34% v 48.53 ± 14.34%; P <.05). Interestingly, at late acute rejection, activated CD4+ and CD8+ T cells remained at almost the same low levels as those on postoperative day 7 in the FK506-treated group. The spleen to body weight ratio was significantly increased in the untreated group (0.53 ± 0.07), and slightly increased in the FK treated group (0.27 ± 0.07, on postoperative day 7; 0.24 ± 0.07 at late acute rejection) compared with the syngeneic group (0.18 ± 0.02). Conclusion: The activation of CD4+ and CD8+ T cells was suppressed effectively by early potent immunosuppressive treatment resulting in prolonged survival of intestinal allograft. At late acute rejection, the CD4+ and CD8+ T cells remained at low-level activation status, in contrast to the surge of CD4+ and CD8+ activation during early acute rejection. This suggests that persistent T cell activation even at low level is sufficient to cause the late acute rejection eventually. A therapeutic strategy targeting these cells is needed for long-term engraftment. J Pediatr Surg 36:352-356. Copyright © 2001 by W.B. Saunders Company.

Preventive effect of FK 506 (tacrolimus hydrate) on experimentally induced acute liver injury in rats.

The aim of the study was to investigate the effect of the immunosuppressant FK 506 (tacrolimus hydrate) on acute liver injury induced by Propionibacterium acnes and lipopolysaccharide (LPS). Acute liver injury was induced in male Wistar rats by injecting the animals with P. acnes (10 mg/rat), and administering LPS (10 microg/rat) seven days later. One group was given FK 506 (1 mg/kg) 24 and 2 hr before administration of LPS, and the other group was given the same dose of saline. The 24-hr survival rate, serum alanine aminotransferase (ALT) concentration, and tumor necrosis factor (TNF) -alpha mRNA and protein concentrations in the liver and spleen were then compared. Hepatic macrophages were also isolated from rats seven days after P. acnes injection, LPS, and FK 506 or saline were added to the culture supernatant, and TNF-alpha production was studied. The 24-hr survival rate was 100% in the FK 506-treated group, in contrast with 16.6% in the saline group. Four hours after LPS injection, the serum ALT concentration was 755 +/- 401 in the saline group versus 119 +/- 42 units/ml (P < 0.01) in the FK 506-treated group. The serum TNF-alpha concentration was lower in the FK 506-treated group (1,419 +/- 957 pg/ml) than in the saline group (9205 +/- 2215) (P < 0.01). The mRNA and protein concentrations in the liver and spleen in the two groups did not differ significantly 1 hr after LPS injection but were significantly lower in the FK 506-treated group after 4 hr. FK 506 did not directly inhibit TNF-alpha production by isolated cultured hepatic macrophages. FK 506 is unable to inhibit initial TNF-alpha production by hepatic macrophages (or probably that by splenic macrophages either) stimulated by injection of LPS in P. acnes + LPS-induced acute liver injury. However, the immunosuppressant does limit hepatic damage by inhibiting subsequent aggravation of inflammation by the cytokine network.

Involvement of neutrophils in ischemic injury. I. Biochemical and histopathological investigation of the effect of FK506 on dorsal skin flaps in rats.

The purpose of this study was to investigate the role of neutrophils in ischemic tissue injury and the possible inhibition by pretreatment with FK506, a neutrophilic modulating agent. A dorsal caudally based skin flap (3 x 9 cm) was used as an ischemic injury model in experimental groups. Prior to flap elevation, FK506 at doses of 0.3 mg per kilogram (group 2), 0.5 mg per kilogram (group 3), and 1.0 mg per kilogram (group 4) was given for 3 days intramuscularly. The relationship among neutrophil accumulation (histopathologically), myeloperoxidase (MPO) activity, malondialdehyde (MDA) content (biochemically) of the flap tissue, and flap survival were studied. Skin flaps showed reduced necrosis in the FK506-treated groups (p < 0.08, p < 0.0001, and p < 0.0001 respectively). The increase in accumulation of neutrophils, and MDA and MPO levels (which were induced by ischemia) observed 1 and 24 hours after flap elevation was diminished by FK506 pretreatment. The increased neutrophilic infiltration, and raised tissue MDA content and MPO activity revealed involvement of both free radical production and neutrophils in ischemia. This injury was decreased by FK506, probably by inhibition of neutrophilic chemotaxis, infiltration, and releasing factors.

Mechanism of concordant corneal xenograft rejection in mice: synergistic effects of anti-leukocyte function-associated antigen-1 monoclonal antibody and FK506.

The mechanisms of corneal xenogeneic immunoreaction, as well as the potential role of immunosuppressive therapy in the suppression of corneal xenograft rejection, have not been thoroughly explored. BALB/c mice who received orthotopic corneal transplants (Lewis rats donors) were administered intraperitoneally anti-leukocyte function associated antigen-1 (LFA-1) monoclonal antibody (mAb) or FK506 (3 mg/kg/day) or both of these immunosuppressants during a 12-day postoperative period. Histological (hematoxylin-eosin stain) and immunohistochemical evaluations of enucleated eyes were performed. Humoral immune response and delayed-type hypersensitivity (ear-swelling assay) were evaluated. The mean (+/-SD) graft survival time in the untreated control, FK506-treated, anti-LFA-1 mAb-treated, and combined-treatment groups was 5.8+/-0.8, 9.4+/-4.0, 8.7+/-5.0, and 67.7+/-16.4 days, respectively. In the untreated control group, mouse IgG, IgM, and C3 were expressed on the rat corneal grafts during the early postoperative phase. Flow cytometry studies revealed high titers of xenoreactive IgG and IgM antibodies. T helper 1 cytokines were expressed on xenografted corneal beds, and delayed-type hypersensitivity was induced. However, local expression of IgM, C3 and T helper 1 cytokines, serum antibodies of IgG and IgM, and delayed-type hypersensitivity were suppressed in the anti-LFA-1 mAb- plus FK506-treated group. Both humoral and cell-mediated immune reaction play an important role in the initial rejection in rat-to-mouse corneal xenotransplantation. The treatment with anti-LFA-1 mAb in combination with FK506 synergistically suppresses concordant corneal xenogeneic reaction.

Allogeneic hepatocyte and fetal liver transplantation and xenogeneic hepatocyte transplantation for nagase's analbuminemic rats

Allogeneic hepatocyte and fetal liver transplantation and xenogeneic hepatocyte transplantation were performed in Nagase's analbuminemic (NAR, RT1 1) rats, and the functional and morphological changes of intrasplenic grafts under FK506 immunosuppression were examined. Normal male Wistar Shi rats (RT1 k) and male BALB/C mice were used as donors. Serum albumin levels in FK506-treated NAR rats 4 wk after all three transplantation techniques performed were significantly higher than those in animals without immunosuppression, indicating the intrasplenic grafts had survived in FK506-treated groups. On histological examination, hepatocyte colonies were detected in FK506-treated rats after allogeneic hepatocyte or fetal liver transplantation. NAR rats are expected to be useful for studies on allogeneic or xenogeneic hepatocyte transplantation under immunosuppression by various methods.