Long-term host unresponsiveness to encapsulated xenogeneic myoblasts after transient immunosuppression

BACKGROUND: Encapsulating cells prevents the immune destruction of allogeneic cells in the subcutaneous site as well as allogeneic and xenogeneic cells in the central nervous system. However, when encapsulated xenogeneic cells are implanted s.c., they may be subject to rejection by the host. METHODS: Murine C2C12 myoblasts engineered to secrete mouse erythropoietin (mEpo) were used to evaluate the response of control versus FK506-treated xenogeneic recipients (Fischer rats) to encapsulated myoblasts implanted in the s.c. site. RESULTS: Encapsulated C2C12 mEpo cells were rapidly eliminated in immunocompetent Fischer rats. Devices transplanted into nude rats induced a sustained increase in the hematocrit, associated with an extended viability of the encapsulated cells. Short-term immunosuppression with FK506, for periods lasting either 1, 2, or 4 weeks after implantation, permitted the long-term survival of encapsulated C2C12 mEpo cells in Fischer rats. Animals increased their hematocrits to more than 70% and maintained these levels for 13 weeks, independent of the duration of FK506 treatment. Unencapsulated C2C12 mEpo cells injected i.m. in immunosuppressed animals were rejected over this same period. CONCLUSIONS: Encapsulation alone cannot protect xenogeneic myoblasts from immune destruction in the s.c. site. These results highlight the importance of combining the technique of cell encapsulation with transient immunosuppression to achieve long-term survival of xenografted myoblasts in a peripheral immunoreactive site.     

Thymectomy impairs but does not uniformly abrogate long-term acceptance of semi-identical liver allograft in inbred miniature Swine temporarily treated with FK506

BACKGROUND: Long-term acceptance of semi-identical orthotopic liver transplants (OLTs) in inbred swine is induced by a 12-day course of FK506. To study whether acceptance is attributable to central or peripheral immune mechanisms, the effect of complete thymectomy was determined. METHODS: Total thymectomy was performed in 15 swine 3 to 4 weeks before OLT. Twelve of these animals received a 12-day course of FK506 after OLT, and three animals did not receive immunosuppression. Five additional nonthymectomized pigs received OLT and a FK506 regimen. Graft survival, liver function, histology, and cellular and humoral responses were assessed. RESULTS: Nonthymectomized, FK506-treated animals uniformly showed long-term acceptance of OLT and developed stable donor unresponsiveness. Of the 12 thymectomized, FK506-treated pigs, seven died of non-immunologic causes within 3 postoperative months, and five maintained their OLT for more than 6 months (range 180-450 days). Among these survivors, two developed a complete anti-donor response (mixed lymphocyte reaction [MLR], cell-mediated lymphocytotoxicity [CML], and immunoglobulin [IgG] antibodies) and eventually rejected their OLT at postoperative day 180. The three remaining pigs kept their liver allografts up to 450 days and developed a donor-specific unresponsiveness (a transient anti-donor MLR was observed during the follow-up but never an anti-donor CML or IgG antibodies). All three thymectomized, untreated animals rejected their allografts acutely and displayed a complete anti-donor response (MLR, CML, and IgG antibodies). CONCLUSIONS: Complete thymectomy before OLT impaired but did not uniformly abrogate long-term acceptance of semi-identical OLT, suggesting that peripheral immune mechanisms may be sufficient to induce long-term acceptance of liver allografts in some recipients.     

Delivery of erythropoietin by encapsulated myoblasts in a genetic model of severe anemia

BACKGROUND: Existing animal models of anemia inadequately reflect the hematocrit usually present in chronic renal failure (CRF) patients and do not permit long-term treatment studies. The transgenic mouse strain 134.3LC (Epo-TAg(H)) displays a severe chronic anemia resembling that observed clinically during CRF, while displaying an active, normal life span. This phenotype makes it a particularly interesting mouse model for testing erythropoietin (Epo)-based gene transfer strategies. METHODS: Ex vivo gene therapy was employed to administer mouse Epo to homozygous anemic Epo-TAg(H) mice. Encapsulated C(2)C(12) myoblasts genetically engineered to secrete 163 IU mouse Epo/10(6) cells/day were subcutaneously transplanted on the dorsal flank of the mice. Efficacy of delivered Epo was monitored by weekly measurements of animal hematocrit. RESULTS: Most treated homozygous Epo-TAg(H) mice displayed only a transient rise in hematocrit before eventually decreasing to levels as low as 3%. Administering the immunosuppressor anti-CD4+ monoclonal antibody (mAb) to homozygous Epo-TAg(H) mice, beginning at the time of implantation, permitted a rise in hematocrit that remained stable at elevated levels in cases of continued immunosuppression. CONCLUSIONS: Mice having the T antigen insertion in both Epo alleles appeared to develop an immune response to the natural mouse Epo delivered by encapsulated cells. By preventing this reaction using immunosuppression, we demonstrate that encapsulated myoblasts can deliver therapeutic doses of mouse Epo systemically and restore hemopoiesis in a genetic model of severe anemia.     

Feasibility of cryopreserved tracheal xenotransplants with the use of short-course immunosuppression

OBJECTIVE: We evaluated the feasibility of discordant xenotransplantation of the cryopreserved trachea with intermittent immunosuppression to help solve the shortage of donor tracheas. METHODS: Two experiments were performed with heterotopic transplantation models in 14 guinea pigs and 85 rats. So that the minimal dose of FK506 for viable fresh xenografts could be determined, FK506 was given in escalating doses (0, 1.5, 2.5, and 3.5 mg/kg) for recipient animals after xenogeneic transplantation. With the goal of obtaining a long-term survival of the xenografts, the effect of cryopreservation on xenografts was assessed and thereafter different cycles of immunosuppression every third week were evaluated in fresh or cryopreserved xenografts in the second experiment. RESULTS: An FK506 dosage of more than 2.5 mg/kg per day was much more effective than smaller dosages, as demonstrated by morphologic assessment. A higher dosage of FK506 potentially delayed the rejection of xenografts and can thus maintain tracheal xenograft viability for less than 4 weeks in rat recipients. In experiment 2, the cryopreserved xenografts showed less histologic viability than fresh xenografts but greater patency of the lumen. The patency of cryopreserved xenografts was favorably maintained for a longer period than that of fresh xenografts with either the same number or more cycles of immunosuppression. CONCLUSIONS: We conclude that the synergistic effect of cryopreservation and adequate intermittent immunosuppression may enable tracheal xenografts to remain viable over longer periods.     

A 12-day course of FK506 allows long-term acceptance of semi-identical liver allograft in inbred miniature swine

BACKGROUND: Spontaneous tolerance to liver allograft has been reported previously in outbred pig models, but the lack of genetic background did not allow to analyze the impact of the major histocompatibility complex (MHC) on tolerance induction. A model of semi-identical liver allograft was therefore developed in inbred miniature swine in order to mimic the clinical situation of living related liver transplant (parent into infant) and to study a protocol for inducing tolerance to liver allograft. METHODS: SLAdd (class Id/d, class IId/d) pigs received orthotopic liver allograft from heterozygous SLAcd (class Ic/d, class IIc/d) miniature swine. Eight animals did not receive immunosuppression. Fourteen SLAdd animals had a 12-day course of FK506 and were divided in two subgroups. In subgroup FK-1, six pigs received a daily intramuscular injection of FK506 at 0.1-0.4 mg/kg, in order to reach daily trough levels between 7 and 20 ng/ml; in subgroup FK-2, eight additional animals received two daily injections of FK506 at 0.05 mg/kg regardless of the daily trough levels. Graft survival, liver biological tests, histology, cellular and humoral immune responses, as well as detection of microchimerism were assessed in all groups. RESULTS: All untreated animals rejected their allograft and died within 28.1 +/- 9.5 days. These rejector animals developed a significant anti-donor cellular and humoral immune response. No peripheral or lymphoid tissue microchimerism was detected in this group. In contrast, long-term survival was obtained in five FK-treated animals (112, 154, 406, 413, and 440 days), whereas several pigs died with a normal allograft function from either overimmunosuppression or intercurrent causes. All FK-treated pigs developed a specific anti-donor unresponsiveness in both cell mediated lymphocytotoxicity and mixed lymphocyte reaction and did not develop anti-donor alloantibodies. The study of the anti-donor immune response by mixed lymphocyte reaction, during the first postoperative week, demonstrated a specific anti-donor unresponsiveness in the peripheral blood from the first posttransplant day. Although microchimerism was detectable in the peripheral blood for several postoperative weeks (maximum 10 weeks) in FK-treated animals, donor cells or DNA were not detected during the long-term follow-up in peripheral blood or lymphoid tissues. CONCLUSIONS: Spontaneous tolerance to semi-identical orthotopic liver allograft did not occur, whereas a 12-day course of FK506 allowed long-term graft acceptance. All FK-treated animals developed in vitro signs of specific immune unresponsiveness and transient peripheral microchimerism. The specific anti-donor cellular unresponsiveness occurred on the first postoperative day after surgery and was of long-term duration. The study of the early immunological events in this model could be of major importance regarding clinical living related liver transplantation.     

Chimeric Allografts Induced by Short‐Term Treatment With Stem Cell–Mobilizing Agents Result in Long‐Term Kidney Transplant Survival Without Immunosuppression: A Study in Rats

Transplant tolerance allowing the elimination of lifelong immunosuppression has been the goal of research for 60 years. The induction of mixed chimerism has shown promise and has been extended successfully to large animals and to the clinic; however, it remains cumbersome and requires heavy early immunosuppression. In this study, we reported that four injections of AMD3100, a CXCR4 antagonist, plus eight injections of low‐dose FK506 (0.05 mg/kg per day) in the first week after kidney transplantation extended survival, but death from renal failure occurred at 30–90 days. Repeating the same course of AMD3100 and FK506 at 1, 2 and 3 mo after transplant resulted in 92% allograft acceptance (n = 12) at 7 mo, normal kidney function and histology with no further treatment. Transplant acceptance was associated with the influx of host stem cells, resulting in a hybrid kidney and a modulated host immune response. Confirmation of these results could initiate a paradigm shift in posttransplant therapy.     

Effect of DSG on xenogeneic immune reactivity with special emphasis on human anti-pig cellular reactions in vitro

Abstract: The effect of the immunosuppressive drug 15-deoxyspergualin (DSG) on xenogeneic human anti-porcine cellular reactivity in vitro, including MLR induced proliferation, interleukin-2 (II-2) production, generation of cytotoxic cells, and the effect on antibody-dependent cellular cytotoxicity (ADCC), were compared with the effects of cyclosporin A (CsA) and/or FK506. The cytotoxic response was evaluated for both direct and indirect pathways for antigen presentation. In addition, the effects of DSG and CsA on antibody production to pig peripheral blood lymphocytes (PBL) in mice was studied. The degree of immunosuppression of xenogeneic and allogeneic cellular responses was compared. CsA and FK506 effectively inhibited proliferation and II-2 production induced by allogeneic human PBL or xenogeneic porcine PBL, whereas DSG did not have any effect on these responses. However, DSG suppressed both the allogeneic and xenogeneic in vitro induced cytotoxic responses, to the same level whether induced via the direct or indirect pathways of immune activation. In contrast, CsA inhibited cytotoxicity induced by xenogeneic cells via the direct but not via the indirect pathway. No effect of FK506 and DSG on ADCC was demonstrated.
A 5-day treatment with DSG or CsA of mice immunized with pig PBL partly suppressed antibody production. In DSG treated mice anti-pig PBL antibodies were produced, but titers were lower than in nontreated or CsA treated mice. The results indicate that DSG may be more effective than CsA/FK506 in inhibiting cytotoxic responses and antibody production induced by xenogeneic pig cells. A possible explanation could be that cytotoxicity induced via the indirect activation pathway of xenoreactivity is mediated to a high degree by CD3^- CD16⁺ (natural killer) NK-like cells, and that stimulation of these cells may be more sensitive to DSG than to CsA/FK506.     

Combined immunosuppression of mycophenolate mofetil and FK506 for myoblast transplantation in mdx mice

BACKGROUND: Overcoming adverse effects of immunosuppressors can be achieved by combining different drugs, thus allowing a dosage reduction. Myoblast transplantation is a potential therapy for Duchenne muscular dystrophy. Our research group previously established that FK506 (tacrolimus) is an effective immunosuppressive drug for myoblast transplantation in mice and monkeys. METHODS: In the present study, a reduced dose of FK506 at 1.0 mg/kg/day was used in combination with mycophenolate mofetil (MMF; 80 mg/kg/day) as an immunosuppressive protocol for myoblast transplantation. Graft success was evaluated by quantifying the number of dystrophin-positive fibers per muscle section that were injected with normal cells. RESULTS: MMF used alone could not prevent immune rejection of the transplanted myoblasts. MMF given in combination with FK506 immediately after transplantation reduced the success of myoblast transplantation by about 50%. A low dose of FK506 combined with MMF after the establishment of the graft (3 weeks) maintained graft success and controlled immune infiltration compared with a low dose of FK506 alone. However, lymphocyte infiltration was observed at longer term using a low dose of FK506 combined with MMF. CONCLUSIONS: The diminution of graft success when combining FK506 and MMF by the time of myoblast transplantation could be attributed to the inhibition of myoblast fusion by MMF. The use of MMF and FK506 after the establishment of the graft did not reduce graft success, however, this combination was not effective at controlling long-term immune rejection in comparison with the optimal dose of FK506 alone.     

Evaluation of an intrathecal immune response in amyotrophic lateral sclerosis patients implanted with encapsulated genetically engineered xenogeneic cells

A phase I/II clinical trial has been performed in 12 amyotrophic lateral sclerosis (ALS) patients to evaluate the safety and tolerability of intrathecal implants of encapsulated genetically engineered baby hamster kidney (BHK) cells releasing human ciliary neurotrophic factor (CNTF). These patients have been assessed for a possible intrathecal or systemic immune response against the implanted xenogeneic cells. Hundreds of pg CNTF/ml could be detected for several weeks in the cerebrospinal fluid (CSF) of 9 out of 12 patients, in 2 patients up to 20 weeks after capsule implantation. Slightly elevated leukocyte counts were observed in 6 patients. Clear evidence for a delayed humoral immune response was found in the CSF of only 3 patients out of 12 (patients #4, #6, and #10). Characterization of the antigen(s) recognized by the antibodies present in these CSF samples allowed to identify bovine fetuin as the main antigenic component. The defined medium used for maintaining the capsules in vitro before implantation contains bovine fetuin. Fetuin may therefore still be adsorbed to the surface of the cells and/or the polymer membrane, or be present in the medium surrounding the encapsulated cells at the time of implantation. Because of the insufficient availability of CSF samples, as well as the relatively poor sensitivity of the assays used, a weak humoral immune response against components of the implanted cells themselves cannot be excluded. However, the present study demonstrates that encapsulated xenogeneic cells implanted intrathecally can survive for up to 20 weeks in the absence of immunosuppression and that neither CNTF nor the presence of antibodies against bovine fetuin elicit any adverse side effects in the implanted patients.     

Six-month survival of microencapsulated pig islets and alginate biocompatibility in primates: proof of concept

BACKGROUND: Pig islets xenotransplantation remains associated with a strong humoral and cellular xenogeneic immune responses. The aim of this study was to assess the long-term biocompatibility of alginate encapsulated pig islets after transplantation in primates. METHODS: Adult pig islets encapsulated in alginate under optimal conditions (n=7) or not (n=5) were transplanted under the kidney capsule of nondiabetic Cynomolgus maccacus. Additional primates received empty capsules (n=1) and nonencapsulated pig islets (n=2) as controls. Capsule integrity, cellular overgrowth, pig islet survival, porcine C-peptide and anti-pig IgM/IgG antibodies were examined up to 6 months after implantation. RESULTS: Nonencapsulated islets and islets encapsulated in nonoptimal capsules were rapidly destroyed. In seven primates receiving perfectly encapsulated pig islets, part of the islets survived up to 6 months after implantation without immunosuppression. Porcine C-peptide was detected after 1 month in 71% of the animals. The majority of grafts (86%) were intact and completely free of cellular overgrowth or capsule fibrosis. Explanted capsules, after 135 (n=2/2) and 180 (n=2/3) days, demonstrated residual insulin content and responses to glucose challenge (stimulation index of 2.2). Partial islet survival was obtained despite an elicited anti-pig IgG humoral response. CONCLUSIONS: Optimal alginate encapsulation significantly prolonged adult pig islet survival into primates for up to 6 months, even in the presence of antibody response.     

Immunosuppression with either cyclosporine a or FK506 supports survival of transplanted fibroblasts and promotes growth of host axons into the transplant after spinal cord injury

Fibroblasts that have been genetically modified to secrete neurotrophins can stimulate axonal regeneration, rescue injured neurons, and improve function when grafted into a spinal cord injury site. These grafts are usually allografts that require immunosuppression to prevent rejection. In this study, we compared the effects of two immunophilin-ligands (cyclosporine A [CsA] and FK506) that are used clinically to prevent transplant rejection on protection of grafted fibroblasts. As there are risks associated with prolonged immunosuppression, we compared the effects of 2 or 8 weeks of administration of these drugs, in combination with our standard methylprednisolone protocol, in animals that survived for 8 weeks, to determine whether a shorter course of immunosuppression would be effective. Outcome measures included fibroblast survival, infiltration of activated macrophages and microglia into the graft, final lesion size, and growth of host axons into the graft. The graft consisted of a Vitrogen matrix into which fibroblasts were suspended; the graft was placed into a C3/C4 lateral funiculus lesion. The fibroblasts were isolated from a transgenic strain of Fischer rats that produce the marker alkaline phosphatase (Fb/AP). This enabled us to track the grafted fibroblasts and to evaluate the extent of their survival. The grafted matrix filled the lesion cavity. The density of fibroblasts within the matrix differed according to treatment. Fibroblast survival was most robust in animals that received 8 weeks of immunophilin-ligand treatment. FK506 supported greater Fb/AP survival than CsA. ED-1 immunostaining for activated microglia and macrophages showed an inverse correlation between AP immunoreactivity and the density of immune cells within the graft. Thus, prolonged administration of either FK506 or CsA was necessary for maximal fibroblast survival and for limiting the macrophage invasion of the graft. None of the FK506 or CsA protocols modified the size of the lesion, indicating that these immunophilin-ligands had little effect on secondary enlargement of the lesion and therefore little neuroprotective effect. Because immunophilin-ligands have been shown to be neurotrophic, we used RT-97 immunostaining for neurofilaments and calcitonin gene related protein (CGRP) staining for dorsal root axons to visualize axons that grew into the graft. Some axons grew into the matrix even in the absence of immunophilin-ligand treatment, suggesting that the Vitrogen matrix itself is permissive, but all of the immunophilin-ligand protocols were much more effective in eliciting axonal growth. Growth of axons into the transplants was equally increased by drug treatment for 2 or 8 weeks. Thus, both treatments improved fibroblast survival, diminished immune cell invasion, and promoted axonal growth, and a 2-week course of treatment with either immunophilin-ligand was as effective as 8 weeks in stimulating axonal growth.     

Short-course immunosuppression using FK506 for rat tracheal allografts

BACKGROUND: A minimizing immunosuppression after a tracheal allotransplantation is desirable. METHODS: We examined the usefulness of a short-course of immunosuppression after tracheal allotransplantation in rat. Each transplant consisting of a 5-ring segment was heterotopically implanted into the omentum. Four animals underwent a syngeneic transplantation and thus served as controls (Group A). Thirty animals underwent an allogeneic transplantation and were randomly classified into 4 groups as follows: No immunosuppression (Group B, n=6), treatment with 0.5 mg/kg of Tacrolims (FK506) (Group C, n=8), 1.0 mg/kg of FK506 (Group D, n=8), and 1.5 mg/kg of FK506 (Group E, n=8). Different doses of FK506 were administered intramuscularly for only three consecutive days after heterotopic tracheal allotransplantation. The serum levels of FK506 were then investigated 3, 7, 14, 21, and 28 days after transplantation in groups C, D, and E. All rats were killed 28 days after transplantation and then the implanted tracheae were harvested, and evaluated histologically. RESULTS: All animals survived for the protocol period. The graft morphology of Group E was significantly better than that of groups B, C, and D regarding both macro- and microscopy, and also showed the same findings as that of Group A, except for low-grade mononuclear cell infiltration. Only in Group E, the FK506 blood level was maintained at over 0.5 ng/ml, which is the lowest detectable limit in this assay, until 21 days after transplantation. CONCLUSIONS: We thus conclude that 1.5 mg/kg of FK506 which was administered for only three consecutive days after surgery may be used to maintain the morphology of tracheal allografts in rats for 28 days after transplantation.     

Optimization of immunosuppressive therapy for spinal grafting of human spinal stem cells in a rat model of ALS

Previous rodent studies employing monotherapy or combined immunosuppressive regimens have demonstrated a variable degree of spinal xenograft survival in several spinal neurodegenerative models including spinal ischemia, trauma, or amyotrophic lateral sclerosis (ALS). Accordingly, the characterization of optimal immunosuppressive protocols for the specific neurodegenerative model is critical to ensure reliable assessment of potential long-term therapeutic effects associated with cell replacement. In the present study we characterized the survival of human spinal stem cells when grafted into the lumbar spinal cords of a rodent model of ALS, SOD1 (G93A) male and female rats (60-67 days old). Four different immunosuppressive protocols were studied: i) FK506 (q12h); ii) FK506 (qd) + mycophenolate (PO; q12h, up to 7 days postop); iii) FK506 (qd) + mycophenolate (IP; q12h, up to 7 days postop); and iv) FK506 (qd) + mycophenolate (IP; qd, up to 7 days postop). Three weeks after cell grafting the number of surviving human cells was then systematically assessed. The highest density of grafted cells was seen in animals treated with FK506 (qd) and mycophenolate (IP; qd; an average 915 ± 95 grafted cells per spinal cord section). The majority of hNUMA-positive cells colocalized with doublecortin (DCX) immunoreactivity. DCX-positive neurons showed extensive axodendritic sprouting toward surrounding host neurons. In addition, migrating grafted cells were identified up to 500 μm from the graft. In animals treated with FK506 (q12h), FK506 (qd) + mycophenolate (PO; q12h) or FK506 (qd) + mycophenolate (IP; q12h), 11.8 ± 3.4%, 61.2 ± 7.8%, and 99.4 ± 8.9% [expressed as percent of the FK506 (qd) and mycophenolate (IP; qd)] cell survival was seen, respectively. In contrast to animals treated with a combination of FK506 + mycophenolate, robust CD4/8 immunoreactivity was identified in the vicinity of the injection tract in animals treated with FK506 only. These data suggest that a combined, systemically delivered immunosuppression regimen including FK506 and mycophenolate can significantly improve survival of human spinal stem cells after intraspinal transplantation in SOD1 (G93A) rats.     

Donor leukocytes combine with immunosuppressive drug therapy to prolong limb allograft survival

Donor leukocytes administered at the time of transplantation may prolong organ allograft survival. This study examined the effectiveness of donor leukocyte injection combined with immunosuppression for limb transplantation across the strong histocompatibility barrier of a Brown Norway donor to a Lewis recipient. Eight animals received 6 x 10(7) donor leukocytes injected on the day of transplantation. From day 1, FK506 (2 mg/kg/d), mycophenolate mofetil (MMF) (15 mg/kg/d), and prednisone (0.5 mg/kg/d) were administered for 2 weeks. After week 2, prednisone and MMF were both tapered by 20% of the initial dosage per week. After week 7, the animals received only FK506 (2 mg/kg/d). From week 8, FK506 was tapered to the maintenance dose of 0.8 mg/kg/d at week 10 and was stopped on week 24. A control group of 8 animals underwent identical treatment except that the leukocyte injection was omitted. Rejection was observed in both groups during FK506 monotherapy; however, the onset of early rejection episodes was significantly later, the period for reversal of the first rejection was significantly shorter, and the dosage of FK506 at the time of rejection was significantly lower among leukocyte-treated recipients. After completion of immunosuppression, survival was modestly prolonged in the leukocyte-treated group. One animal is surviving without immunosuppression on day 234. This trial of donor leukocyte injection combined with immunosuppression in limb transplantation showed a modest, but significant, improvement in outcome.     

Suppressed splenocyte proliferation following a xenogeneic skin graft due to implanted biomaterials

BACKGROUND: Immune system responses to antigens in the context of biomaterials are poorly understood. Biomaterial implantation results in an inflammatory reaction, which is anticipated to alter the adaptive immune response, in the case presented here, to a skin xenograft. Our earlier work showed unexpectedly low splenocyte proliferation following a xenogeneic cell implant in tandem with a biomaterial in the form of a microcapsule. Here we explore whether that effect was due to the cells or the biomaterial, and attempt to dissect the mechanism of immune deviation. METHODS: We assessed the immune response of Balb/c mice to hamster skin grafts accompanied by one of three implants: encapsulated xenogeneic cells; free cells accompanied by the same encapsulation biomaterials; and the encapsulation biomaterials without cells. Cells were encapsulated in a hydroxyethyl methacrylate-methyl methacrylate copolymer then embedded in an agarose gel. Splenocyte proliferation upon re-challenge in vitro, antibody titer in serum, and Th1/2 polarization (by cytokines in splenocyte challenge supernatants and antibody isotypes in serum) were measured. RESULTS: All skin grafts with encapsulation materials (even without cells) suppressed subsequent splenocyte proliferation at 10 days postimplant, although this effect disappeared by two months. In contrast, the antibody response was equal to or greater than that for a skin graft alone. Th1/2 polarization could not explain these observations because it did not correlate with the suppression of splenocyte proliferation. CONCLUSIONS: Implanted biomaterials caused nonspecific, transient suppression of splenocyte responses to hamster cells following hamster skin grafts, which is potentially important in the context of tissue engineering.     

肝移植术后长期存活慢性肾功能损害受者的免疫抑制方案个体化应用

目的探讨肝移植术后长期存活慢性肾功能损害受者应用个体化免疫抑制方案的疗效。方法选择18岁以上、肝移植术后2年以上、入组前采用以他克莫司(FK506)为基础免疫抑制方案、肝功能正常而肾功能损害的受者,共32例。根据免疫功能评分和白细胞计数制定个体化免疫抑制方案,以FK06用量最小化为原则,转换为麦考酚吗乙酯(MMF)或西罗莫司,并调整其用量。调整后至少每个月随访1次,进行肝功能、肾功能、血常规检查和免疫功能评估。结果 32例受者经个体化免疫抑制方案治疗,随访(24.3±7.6)个月,个体化治疗后各时段的肾小球滤过率(GFR)均较此前有明显提高(均为P<0.01),以调整用药后1个月最明显。无发生排斥反应。结论根据免疫功能评分和白细胞计数制定个体化免疫方案,使FK506用量最小化,可以有效改善肝移植术后长期存活的受者的肾功能,并不增加排斥反应的发生率。     

FK506 rescues peripheral nerve allografts in acute rejection

This study investigated the ability of the immunosuppressant FK506 to reverse nerve allograft rejection in progress. Eighty-four Buffalo rats received posterior tibial nerve grafts from either Lewis or Buffalo donor animals. Allografts were left untreated for either 7, 10, or 14 days before receiving daily subcutaneous FK506 injections (2 mg/kg). Time-matched control animals received either an isograft, an allograft with continuous FK506, or an allograft with no postoperative FK506 therapy. All animals underwent weekly evaluation of nerve function by walking track analysis. Experimental group animals were sacrificed either immediately prior to initiation of FK506 therapy (days 7, 10, or 14), after 2 weeks of immunosuppressive treatment, or 8 weeks postsurgery. Histomorphometric analysis, consisting of measurements of total number of nerve fibers, neural density, and percent of neural debris, demonstrated a statistically significant increase in regeneration in the isograft group relative to the untreated allograft group within 28 days of transplantation. Grafts harvested from animals receiving 2 weeks of FK506 after 7 or 10 days of rejection were histomorphometrically similar to time-matched isografts. By contrast, grafts from animals receiving 2 weeks of FK506 following 14 days without therapy resembled untreated allografts and demonstrated significant histomorphometric differences from isografts at the corresponding time point. Analysis of walking track data confirmed that relative to untreated allografts, functional recovery was hastened in animals receiving an isograft, or allograft treated with FK506. This study demonstrated that when started within 10 days of graft placement, FK506 could reverse nerve allograft rejection in rats evaluated following 2 weeks of treatment.     

Neuroimmunophilin ligands improve functional recovery and increase axonal growth after spinal cord hemisection in rats

We have previously shown that FK506 accelerates the rate of nerve regeneration in the peripheral nervous system (PNS) and increases regeneration of central nervous system (CNS) axons into a peripheral nerve graft. In the present study, we examined whether FK506 and a nonimmunosuppressive derivative (FK1706) improve functional recovery and long distance regeneration following a hemisection lesion of spinal cord at T10/T11. Rats were given daily subcutaneous injections of either FK506 (2 mg/kg/day), FK1706 (2 mg/kg/day), an equivalent volume of saline or 30% DMSO as vehicle, respectively. Functional recovery was assessed using a modified Tarlov/Klinger scale, walking along progressively narrower wooden beams (7.7-1.7 cm widths), and analysis of footprints obtained during walking. Compared to both control groups, FK506 and FK1706-treated animals demonstrated significant functional recovery 4 days (beam walking), 2 weeks (footprints), and 4 weeks (Tarlov/Klinger scale). By 11 weeks, FK506-treated and FK1706-treated animals were able to walk, albeit poorly, along even the narrowest (1.7 cm) beam. At 11 weeks, the spinal cords were re-exposed and a small piece of gel foam-soaked Fluoro-Gold was placed on the injured side 2-cm caudal to the first injury. Five days later, the animals were perfused and tissues prepared for fluorescence microscopy. FK506-treated and FK1706-treated rats demonstrate a significantly greater number of retrogradely labeled neurons in the red nucleus. The results implicate a nonimmunosuppressant mechanism in FK506's action and suggest that FK506 or a nonimmunosuppressant derivative may be useful for treatment of spinal cord injuries.     

Control of inflammatory damage by anti-LFA-l: Increase success of myoblast transplantation

Myoblast transplantation is a potential treatment for Duchenne Muscular Dystrophy. This article confirms by experiments in mice that one problem that has limited the success of clinical trials of this procedure is a rapid (within 3 days) inflammatory reaction which kills most of the injected myoblasts. The death of the transplanted myoblasts can be prevented by treating the host with a mAb against LFA-1. This led to a 27-fold increase in the number of muscle fibers expressing a reporter gene present in the donor myoblasts when the host is also adequately immunosuppressed with FK506. Therefore, both the nonspecific inflammatory reaction and the specific immune response should be adequately controlled following myoblast transplantation.     

Effect of FK506 Ointment (Protopic) on Rat Skin Allograft Model

Objective

FK-506 (tacrolimus) is a well known immunosuppressive agent used to prevent allograft rejection. The need for chronic allograft immunosuppression and the consequent harmful systemic effects preclude the use of tissue allograft as a routine surgical reconstructive option. This study assessed the effects of FK-506 ointment (Protopic) therapy versus subcutaneous injection of FK-506 (Prograf) on rat skin graft model.

Methods

Donor Wistar rat dorsal skin was grafted to recipient Sprague-Dawley rats. Animals groups were divided into 2 groups: Group I was treated with intravenous injection of FK-506, and group II was treated with FK-506 ointment for 2 weeks after surgery. Graft appearance challenges were assessed.

Results

FK506 ointment could prolong the median allograft survival time (16.7 days) compared with group I (15.8). Hematoxylin-eosin staining performed on the allo-skin biopsy samples obtained from both group I and II animals at 2 weeks after graft revealed moderate degree of skin rejection accompanied by mixed lymphocyte infiltration. Tacrolimus mean blood levels were much lower in group II (<0.2 ng/mL) than in group I (0.45 ng/mL)

Conclusions

Immunosuppressive FK506 ointment therapy has similar effect to intravenous injection and it could be a useful therapy in the prevention of skin allograft rejection.