Short-term Immunosuppression Research Articles

Long‐term reversal of hypercholesterolemia in low density lipoprotein receptor (LDLR)‐deficient mice by adenovirus‐mediated LDLR gene transfer combined with CD154 blockade

Background Deficiency of the low density lipoprotein receptor (LDLR) results in abnormal elevation of cholesterol within the intermediate and low density plasma lipoproteins (IDL/LDL), and predisposes to early onset atherosclerosis. Cholesterol reduction after adenovirus-mediated LDLR gene transfer to LDLR-deficient animals is transient, due to the elicitation of an immune response. Methods The LDLR-deficient mouse was used as a model to investigate adenovirus-mediated LDLR gene transfer combined with short-term immunosuppression as a cholesterol lowering therapy. Mice were infused with replication-deficient recombinant adenovirus encoding LDLR under control of the cytomegalovirus promoter/enhancer (AdLDLR), and injected with a blocking antibody directed against CD154 (CD40 ligand) to suppress immune responses against the vector and foreign transgene product. Results Mice given AdLDLR and treated with anti-CD154 expressed LDLR on hepatocytes and maintained cholesterol levels below or within normal range for at least 92 days. In contrast, without adjunct immunosuppression LDLR expression was transient, corresponding to temporary decline in cholesterol levels. Analysis of cholesterol concentrations in fractionated plasma showed remarkable reduction in all lipoprotein fractions at early time-points after gene transfer. At later time-points, non-immunosuppressed control mice regained the disease profile with elevated IDL/LDL cholesterol, while profiles of anti-CD154-treated mice were similar to normal. LDLR mRNA transcripts were present in livers of the anti-CD154-treated mice but not controls, 93 days after AdLDLR injection. However, vector DNA was detected in livers of both groups. These results suggest that loss of LDLR expression in the non anti-CD154-treated mice was in part due to immune-mediated promoter silencing, and that anti-CD154 prevented this effect. Conclusion Treatment with anti-CD154 antibody inhibits immune-mediated loss of transgene expression, enabling long-term reduction in cholesterol levels after AdLDLR gene transfer to LDLR-deficient mice. Copyright © 2000 John Wiley & Sons, Ltd.

Long-term reversal of hypercholesterolemia in low density lipoprotein receptor (LDLR)-deficient mice by adenovirus-mediated LDLR gene transfer combined with CD154 blockade.

Deficiency of the low density lipoprotein receptor (LDLR) results in abnormal elevation of cholesterol within the intermediate and low density plasma lipoproteins (IDL/LDL), and predisposes to early onset atherosclerosis. Cholesterol reduction after adenovirus-mediated LDLR gene transfer to LDLR-deficient animals is transient, due to the elicitation of an immune response. The LDLR-deficient mouse was used as a model to investigate adenovirus-mediated LDLR gene transfer combined with short-term immunosuppression as a cholesterol lowering therapy. Mice were infused with replication-deficient recombinant adenovirus encoding LDLR under control of the cytomegalovirus promoter/enhancer (AdLDLR), and injected with a blocking antibody directed against CD154 (CD40 ligand) to suppress immune responses against the vector and foreign transgene product. Mice given AdLDLR and treated with anti-CD154 expressed LDLR on hepatocytes and maintained cholesterol levels below or within normal range for at least 92 days. In contrast, without adjunct immunosuppression LDLR expression was transient, corresponding to temporary decline in cholesterol levels. Analysis of cholesterol concentrations in fractionated plasma showed remarkable reduction in all lipoprotein fractions at early time-points after gene transfer. At later time-points, non-immunosuppressed control mice regained the disease profile with elevated IDL/LDL cholesterol, while profiles of anti-CD154-treated mice were similar to normal. LDLR mRNA transcripts were present in livers of the anti-CD154-treated mice but not controls, 93 days after AdLDLR injection. However, vector DNA was detected in livers of both groups. These results suggest that loss of LDLR expression in the non anti-CD154-treated mice was in part due to immune-mediated promoter silencing, and that anti-CD154 prevented this effect. Treatment with anti-CD154 antibody inhibits immune-mediated loss of transgene expression, enabling long-term reduction in cholesterol levels after AdLDLR gene transfer to LDLR-deficient mice.

Skin Allograft Survival Following Intrathymic Injection of Donor Bone Marrow

Background. Success has been reported using intrathymic injection in the preconditioning regimen to induce allograft tolerance. Although long-term stable tolerance has been achieved in numerous rodent vascularized solid organ allograft models, tolerance to skin transplants has only been achieved across minor antigenic or concordant species disparities. This study sought to induce tolerance across an allogeneic barrier in a rat model with a major genetic disparity.Materials and methods. Lewis rats were injected intrathymically with 1 × 108 Brown-Norway (BN) bone marrow cells and intraperitoneally with 1.0 cc of rabbit anti-rat anti-lymphocyte serum (ALS). Twenty-one days later, BN skin grafts were placed on the injected animals. Control groups were included to isolate the effect of technique, thymic manipulation, strain specificity, and ALS.Results. Animals receiving both intrathymic bone marrow cells and ALS had a skin graft median survival time of 24 days versus 8 days for the control group (P = 0.003). Groups receiving anti-lymphocyte serum alone or intrathymic bone marrow cell injection alone exhibited no skin graft survival prolongation. Mixed lymphocyte reactions revealed normal responsiveness of tolerant animal lymphocytes to donor strain lymphocytes.Conclusion. This protocol utilizing the intrathymic injection of donor bone marrow cells along with short-term immunosuppression with anti-lymphocyte serum produced markedly prolonged survival of skin allografts transplanted across a major histocompatibility barrier. Although tolerance was incomplete, significant prolongation has not previously been reported in genetic disparities of this degree. These results suggest that the application of this technique for central immune modulation may be beneficial for allograft tolerance induction and deserves further study in large animals models.

Microvascular reanastomozed allogenous iliac crest transplants for the reconstruction of bony defects of the mandible in miniature pigs

The effects of immunosuppression with cyclosporin A and prednisolone regimens for allogenous iliac crest bone grafts used for mandibular reconstruction were investigated in 40 miniature pigs, for periods of 2, 4 and 16 weeks. Autogenous and allogenous bone grafts without immunosuppression served as controls. Specimens were evaluated by routine histology, direct magnified radiography and fluorescence microradiography. Four out of five autogenous transplants showed a preserved vascular architecture and bony union. None of the allogenous transplants without immunosuppression survived. Primary bone healing of the allografts was noted after short-term immunosuppression. However, occlussion of the nutrient vessels was noted ten days postoperatively. The allografts were not rejected after cessation of the immunosuppressive therapy within an observation period of 4–12 weeks. Revascularization of all areas of the allografts and creeping substitution of the transplanted bone were noted after seven weeks. Infection of the allografts, with failure of bony union, was noted in nine animals, but primary healing of allografts with short-term immunosuppression was demonstrated.

Short-Term Outcome of Chronic Immunosuppression on the Development of Breast Lesions in Premenopausal Heart and Lung Transplant Patients

The risk of development of breast lesions in patients on chronic immunosuppression is unknown. In order to assess this risk, a retrospective review was performed of the records of 87 women between the ages of 12 and 47 years who received thoracic organ transplant from 1987 to 1996 at our institution. Inclusion criteria consisted of patients who were premenopausal, had no previous history of breast disease, and survived for at least 1 year posttransplantation. All patients were on a triple immunosuppressive regimen consisting of cyclosporine, steroids, and azathioprine. Mean follow-up was 4 ± 1.2 years with a range of 1–6 years. During this period, 21 patients (24%) with a mean age of 38 ± 10 years had screening or diagnostic mammography. The remainder of patients with a mean age of 24 ± 9 years were followed clinically. Overall, 10 patients (11%) developed a total of 17 palpable, solid lesions at 33 to 72 months posttransplantation. Fifteen of these lesions were surgically excised. Five of the patients had multiple lesions. Pathological examination of the specimens revealed fibroadenoma in nine, fibrocystic disease in four, low grade phylloides tumor in one, and T-cell lymphoma in one case. None of the patients have developed primary breast cancer during follow-up. In conclusion, short-term immunosuppression does not increase the risk of the development of benign breast lesions in young women after thoracic organ transplantation, but rather the distribution of benign lesions is similar in an age-matched population. There were several cases of multiple fibroadenomas in the transplant population, but mammography revealed no malignant disease in this age group and does not need to be utilized in this population beyond what is considered standard for immunocompetent patients. The long-term effect ofimmunosuppressive therapy on the developmentof breast cancer in this group remains to be defined.

IMPROVED OUTCOME AND STEROID WITHDRAWAL IN MYCOPHENOLATE MOFETIL TREATED PRIMARY CADAVERIC RENAL TRANSPLANT RECIPIENTS

232 The long term of corticosteroids in renal transplant recipients continues to be associated with significant morbidity even in the Cyclosporine (CSA) era. Although steroid withdrawal (STW) utilizing maintenance therapy with CSA/azathioprine (AZA) results in a reduction in steroid complications, an increased risk of acute rejection in the early withdrawal period and a gradual but significant increase in the serum creatinine during long term followup was noted. The substitution of Mycophenolate Mofetil (MMF) for AZA has lead to an overall reduction in the number and severity of acute rejection episodes. We report the short term outcome of STW in 2 groups: 18 CSA/MMF treated primary cadaveric renal transplant recipients and 95 CSA/AZA treated patients. STW was initiated in both groups as 9 months posttransplant when the maintenance methylprednisolone dose was 10 mg/day. A decrease of 2 mg/month was started until complete STW. Inclusion criteria for STW required a serum creatinine < 2.5 mg/dl and no prior steroid resistant rejections requiring antibody therapy. MMF was dosed at 1 g twice daily and AZA was provided at 1.5 mg/kg/day. Mean followup was 13 ± 8 months in the CSA/MMF group and 64 ± 32 months in the CSA/AZA group (p < 0.05). STW was accomplished at the same time posttransplant in both groups (CSA/MMF - 14± 2 months vs. 14 ± 3 months for CSA/AZA, p=ns). The incidence of prior steroid sensitive rejection episodes was lower in the CSA/MMF group - 12% vs. CSA/AZA group - 25% (p < 0.05). The 1 and 2 year success of maintaining complete STW was significantly higher and the incidence of acute rejection after STW and recurrent glomerulonephritis (RCR GN) was lower in the CSA/MMF group. TableThe serum creatinine was slightly but not significantly lower in the CSA/MMF group at 1 year (1.5 ± 0.2 mg/dl vs. 1.7 ± 0.2 mg/dl-CSA/AZA) and 2 years (1.5 ± 0.2 mg/dl vs. 1.8 ± 0.3 mg/dl- CSA/AZA). In conclusion, the combined use of CSA/MMF after STW resulted in improved 1 and 2 year success rates with a reduction in the risk of acute rejection after STW compared to a CSA/AZA based protocol. A reduction in early posttransplant rejection episodes may play a role in this improved outcome. MMF appears to add more potent short and long term immunosuppression which may increase the opportunity to achieve successful STW.

AN IMPROVED TECHNIQUE FOR ISOLATING PIG ISLETS: THE IMPORTANCE OF TENSEGRITY IN ISLET LONG TERM CULTURE VIABILITY

176 Pig Islets (PI) are clearly the xenogeneic donor islet of choice for human islet transplant. Pig insulin is known to work well in the human without antibody problems. Furthermore, newer short-term immunosuppression can markedly reduce early graft loss with PI and tolerance induction can even be demonstrated in some discordant xenografts. Isolation of PI remains a major problem with islet fragmentation and islet loss during digestion. In addition, PI undergo a high rate of apoptosis following isolation in culture which can result in 50-95% loss of PI in 24-48 hours. We initially observed that PI embedded in extracellular adherent matrix exhibited an Anchorage-Dependent cell growth and survival with viability of >80%(Ethidium Bromide/Acridine Orange Assay - EB/AO) at 20 days (p<0.01 with controls). We tested the so-called "Tensegrity Concept" that cell viability and growth in culture depends on adhesion and extracellular integrin structuring and "scaffolding". Tensegrity is optimal when the cell cytoskeleton and the extracellular matrix integrate to achieve a physiological balance of intra and extracellular mechanical tension. By shortening collagenase digestion to yield PI attached to acinar matrix cell remnants, we sought to provide a potential extracellular structure to develop tensegrity. Control PI digestions showed 95% purity with pure islets free of attached acinar and extracellular matrix elements, but a loss of EB/AO viability to 40% and 10% in 24 and 48 hours. In contrast, the matrix-embedded PI had a purity of only 52% but an EB/AO viability of 93% and 86% at 24 and 48 hours and 65% at 30-35 days(p<0.05) At 1 month, the acinar tissue had largely died but fibrous strands remained attached to the cells. Islet function in static incubation was excellent with a insulin rise of 420% in response to a increased glucose incubation solution (normal for fresh islets=320%,p=>0.10). Thus, incomplete digestion of PI with retention of attached elements of the acinar and extracellular matrices apparently supplies a tensegrity (extracellular matrix scaffolding) which promotes long term growth and viability of PI in culture. We conclude that incomplete digestion of islets and/or imbedding islets in extracellular matrix markedly enhances the viability and long term yield of PI and maybe the technique of choice for PI digestion and preparation of PI for culture and/or transplantation.

Transient immunosuppression with 15-deoxyspergualin prolongs reporter gene expression and reduces humoral immune response after adenoviral gene transfer.

A strong immune response against transgenic cells is one important limitation for long-term expression after adenoviral gene transfer in mammals. Continuous pharmacological immunosuppression has been shown to ameliorate immune reactions and to prolong reporter gene expression. In this study, we explored the effect of short-term immunosuppression for long-term gene expression and its impact on antibody formation. Immunosuppression with FK 506 (1 mg/kg/day), cyclosporin A (20 mg/kg/day) and 15-deoxyspergualin (10 mg/kg/day) was performed in NMRI mice. Expression of the reporter gene human alpha-1-antitrypsin (hAAT) and antibody formation was monitored for 7 months. A 5-day course of 15-deoxyspergualin (15-DSG) markedly slowed the decline of reporter gene expression and a positive effect was still detectable 200 days after gene transfer. At the same time, antibody production was reduced by 50-60%. Continuous treatment with 15-DSG (10 mg/kg twice weekly) led to a further small increase of gene expression but reduced antibody formation by 80-90%. A short course of FK 506 and cyclosporin A (CsA), had conferred a negative effect on gene expression. Both groups showed an even faster reduction in gene expression compared with the control group. The results of this investigation suggest that 15-DSG could serve as an effective supplement for viral gene therapy protocols.

Transient immunosuppression with FK506 permits long-term expression of therapeutic genes introduced into the liver using recombinant adenoviruses in the rat.

The host immune response limits the duration of expression of adenovirally transduced genes and precludes long-term gene expression upon re-administration of the virus. In this study we wished to evaluate whether short-term immunosuppression of the host, at the time of recombinant virus administration, would allow expression of the therapeutic gene product upon virus reinjection. Gunn rats were used as recipients of recombinant adenoviruses expressing human BUGT (Ad-hBUGT) or E. coli beta-galactosidase (Ad-LacZ). Rats were treated with FK506 (1-1.5 mg/kg, per OS daily) for three days beginning 24 hours before each virus injection. Control groups did not receive any immunosuppressant. The serum bilirubin level was reduced from 7.1 +/- 0.75 mg/dL to 2.0 +/- 0.7 mg/dL within two days of viral injection in both FK506 treated and control groups, and then gradually increased in 6 weeks. FK506-treated rats had low or undetectable antibody titers against the recombinant adenovirus and minimal or no cytotoxic T lymphocyte (CTL) response against adenovirus-infected cells. The tolerized rats received two subsequent injections 42 and 98 days after the first injection, which reduced the bilirubin levels again to 2.0 +/- 0.56 and 2.2 +/- 0.61 mg/dL, respectively. In contrast, control rats developed high titer neutralizing antibodies and a CTL response, and their serum bilirubin levels were not reduced following subsequent injections. We conclude that short-term FK506 treatment around the time of virus administration prevents the host immune response, permitting long-term gene therapy by repeated administration of the recombinant virus.

Transient immunosuppression with FK506 permits long-term expression of therapeutic genes introduced into the liver using recombinant adenoviruses in the rat

The host immune response limits the duration of expression of adenovirally transduced genes and precludes long-term gene expression upon re-administration of the virus. In this study we wished to evaluate whether short-term immunosuppression of the host, at the time of recombinant virus administration, would allow expression of the therapeutic gene product upon virus reinjection. Gunn rats were used as recipients of recombinant adenoviruses expressing human BUGT (Ad-hBUGT) or E. coli beta-galactosidase (Ad-LacZ). Rats were treated with FK506 (1-1.5 mg/kg, per OS daily) for three days beginning 24 hours before each virus injection. Control groups did not receive any immunosuppressant. The serum bilirubin level was reduced from 7.1 +/- 0.75 mg/dL to 2.0 +/- 0.7 mg/dL within two days of viral injection in both FK506 treated and control groups, and then gradually increased in 6 weeks. FK506-treated rats had low or undetectable antibody titers against the recombinant adenovirus and minimal or no cytotoxic T lymphocyte (CTL) response against adenovirus-infected cells. The tolerized rats received two subsequent injections 42 and 98 days after the first injection, which reduced the bilirubin levels again to 2.0 +/- 0.56 and 2.2 +/- 0.61 mg/dL, respectively. In contrast, control rats developed high titer neutralizing antibodies and a CTL response, and their serum bilirubin levels were not reduced following subsequent injections. We conclude that short-term FK506 treatment around the time of virus administration prevents the host immune response, permitting long-term gene therapy by repeated administration of the recombinant virus. (Hepatology 1997 Oct;26(4):949-56)

Genetic correction of dystrophin deficiency and skeletal muscle remodeling in adult MDX mouse via transplantation of retroviral producer cells.

Duchenne muscular dystrophy (DMD) is an X-linked, lethal disease caused by mutations of the dystrophin gene. No effective therapy is available, but dystrophin gene transfer to skeletal muscle has been proposed as a treatment for DMD. We have developed a strategy for efficient in vivo gene transfer of dystrophin cDNA into regenerating skeletal muscle. Retroviral producer cells, which release a vector carrying the therapeutically active dystrophin minigene, were mitotically inactivated and transplanted in adult nude/mdx mice. Transplantation of 3 x 10(6) producer cells in a single site of the tibialis anterior muscle resulted in the transduction of between 5.5 and 18% total muscle fibers. The same procedure proved also feasible in immunocompetent mdx mice under short-term pharmacological immunosuppression. Minidystrophin expression was stable for up to 6 mo and led to alpha-sarcoglycan reexpression. Muscle stem cells could be transduced in vivo using this procedure. Transduced dystrophic skeletal muscle showed evidence of active remodeling reminiscent of the genetic normalization process which takes place in female DMD carriers. Overall, these results demonstrate that retroviral-mediated dystrophin gene transfer via transplantation of producer cells is a valid approach towards the long-term goal of gene therapy of DMD.

The genetic basis of myoadenylate deaminase deficiency is heterogeneous

Inosine 5′-monophosphate dehydrogenase (IMPDH) is a target of the immunosuppressant mycophenolic acid (MPA) which is used for the prevention of acute rejection. We evaluated the concentration-dependent effects of MPA and its phenolic (MPAG) and acyl (AcMPAG) glucuronides on IMPDH activity in erythrocytes in an initial and a stable phase.Eight kidney transplant recipients treated with mycophenolate mofetil (MMF) in an initial phase and 104 recipients [56 received MMF (MMF+) and 48 did not (MMF−)] in a stable phase were enrolled.IMPDH activity in erythrocytes was inhibited at the peak plasma concentration of MPA in initial kidney transplant recipients. However, median IMPDH activity in erythrocytes was 1.73 times higher in MMF+ than in MMF− kidney transplant recipients (38.7 to 22.4 nmol/g hemoglobin/h, P < 0.01). The median values of trough plasma concentrations (C12) of MPA, MPAG, and AcMPAG in stable kidney transplant recipients were 2.86, 49.9, and 0.256 μg/ml, respectively. In a multivariate analysis, AcMPAG C12, MPAG C12, MPA C12, serum creatinine, age, lactate dehydrogenase, and serum albumin were significant predictors, accounting for interindividual variability of IMPDH (R2 = 0.537, P = 0.02).IMPDH activity in erythrocytes may be useful indicators of short-term immunosuppression and long-term exposure of MPA.

Immunosuppression augments growth of graft-adherent Staphylococcus epidermidis.

To determine if systemic suppression of host defenses during graft implantation alters the initial adherence and subsequent growth of Staphylococcus epidermidis on vascular prostheses. Dacron grafts 1 cm2 were implanted in the back subcutaneous tissue of Swiss-Webster mice (n = 247), followed by topical inoculation with 2 x 10(7), 2 x 10(5), 2 x 10(3), or 2 x 10(1) colony-forming units of S epidermidis. Half of the mice were immunosuppressed with cyclophosphamide (150 mg/kg intraperitoneally), to achieve a consistent, significant decrease in the white blood cell count and major histocompatibility complex class II (Ia) expression. Control mice received an equal volume of saline solution. Graft bacterial biofilm concentrations were determined at 1 day for adherence and within 2 weeks for bacterial growth, by using sonication and quantitative agar culture. Immunosuppression did not significantly alter the initial adherence of bacteria to vascular grafts. Immunosuppressed animals that were inoculated with 2 x 10(7) and 2 x 10(5) colony-forming units of S epidermidis had significantly higher bacterial biofilm concentrations as compared with those in control animals. Graft infection persisted at 14 days in all animals, with and without immunosuppression. Suppression of immune function during graft implantation augmented growth of adherent bacteria. The effect of short-term perioperative immunosuppression on late-appearing S epidermidis graft infection needs further study.

Fresh venous allografts in peripheral arterial reconstruction in dogs: Effects of histocompatibility and of short-term immunosuppression with cyclosporine A and mycophenolate mofetil

To date, no arterial substitute has been shown to be as effective as the autologous saphenous vein in peripheral revascularization procedures. In the present study, the venous allograft was evaluated as a vascular substitute in terms of patency and induction of host immune reactivity, whether used in major histocompatibility complex–incompatible, major histocompatibility complex–compatible, or immunosuppressed major histocompatibility complex–incompatible dogs. The immunosuppressive drug therapies were given for a period of 31 days, beginning 1 day before transplantation, and consisted of the use of cyclosporine A, mycophenolate mofetil, or a combination of both. All histoincompatible allografts were thrombosed at 4 or 8 weeks after transplantation with antibody development and cell-mediated cytotoxicity in the graft, whereas histocompatible allografts showed late stenosis without immunologic reactions directed toward donor cells. Given alone, neither cyclosporine A nor mycophenolate mofetil improved the overall patency of venous allografts; thrombosis occurred shortly after cessation of immunosuppression. Still, the cyclosporine A–mycophenolate mofetil combination therapy led to a 100% patency rate at 20 weeks after implantation and immune reactions were markedly reduced. This study shows that the fresh vein allograft is still an attractive and functional alternative to the autologous saphenous vein if the host immunologic reactions are controlled by cyclosporine A–mycophenolate mofetil immunosuppression. (J T HORAC C ARDIOVASC S URG 1995;110:1732-42)

Vascularized Allogeneic Joint, Muscle, and Peripheral Nerve Transplantation

Joint, muscle, and peripheral nerve allotransplantation was done with short-term cyclosporine immunosuppression. To investigate the effectiveness of this regimen, the allografts were examined after withdrawal of cyclosporine. Using inbred rats, vascularized orthotopic allotransplantation of the knee joint, rectus femoris muscle, and great saphenous nerve was done across a major histocompatibility complex barrier. Cyclosporine was administered for 4 to 6 weeks postoperatively, and the grafts were observed until Week 12. Long-term administration of cyclosporine and nonvascularized transplantation were used as controls. Although rejection of the allografts could be delayed for 2 to 3 weeks after the withdrawal of cyclosporine, all transplanted joints, muscles, and nerves eventually were rejected completely and immunotolerance could not be induced. The joint allografts at first achieved bony union, but eventually were destroyed because of pathologic fractures. In the group treated with long-term immunosuppression, the allografts showed no rejection and functional improvement was obtained. However, rats given a high dose (10 mg/kg per day) of cyclosporine died from adverse effects of the drug by Week 12. In the nonvascularized treatment group, the results were poor in every patient, and the need for graft vascularization for skeletal tissue allotransplantation was confirmed.

Immunosuppressive effect of 15-deoxyspergualin applied to peripheral nerve allotransplantation in the rat

15-Deoxyspergualin (15-DSG), which has a unique immunosuppressive action, was applied to peripheral nerve allotransplantation. Its effects on graft survival were experimentally assessed using inbred rats. The sciatic nerve (20 mm) allotransplantation model was created in two different strains. An attempt was made to answer the following two questions: (1) can short-term immunosuppression alone produce sufficient immunological tolerance to maintain graft survival indefinitely? (2) can graft rejection be prevented by chronic intermittent low-dose 15-DSG administration (2.5 mg/kg/day), and to what extent does nerve regeneration occur? To evaluate the efficacy of 15-DSG, a comparison was made with autografts, allografts with no immunosuppression, and allografts immunosuppressed with cyclosporine (CsA), a strong immunosuppressant. The results indicate that short-term 15-DSG therapy is incapable of inducing immunotolerance of peripheral nerve allografts. Because nerve conduction in the rejected allografts was better preserved than in the CsA group, short-course 15-DSG therapy appeared to provide better results than CsA therapy for peripheral nerve allotransplantation.

Vascularized Allogeneic Nerve Transplantation with Cyclosporine Immunosuppression

Allogenic grafts have many advantages over autogenous grafts. An experimental rat model using the great saphenous nerve was developed to study the reconstruction of long peripheral nerve defects with vascularized allogeneic nerve grafts, and the effects of cyclosporine therapy on graft survival were assessed. In animals receiving short-term immunosuppression, the number of regenerated axons increased steadily during the treatment, but rejection began immediately after cyclosporine was discontinued. As the tissues transplanted with the nerves underwent necrosis, the grafts showed demyelination. In contrast, in animals receiving long-term cyclosporine therapy, nerve regeneration assessed in terms of the myelinated axon count was excellent until at least 12 weeks postoperatively but was less active than in vascularized autografts or conventional allografts. These results indicate that the short-term immunosuppressive therapy with cyclosporine cannot produce immunological tolerance of vascularized nerve allografts. In addition, even when administration of cyclosporine was prolonged, nerve regeneration did not exceed that in autografts.

Immunology: Immunosuppression by corticosteroid has no effect on the pregnancy rate in routine in-vitro fertilization/embryo transfer patients

Immunosuppression by exogenous corticosteroids has been used to improve the rates of embryo implantation and pregnancy in in-vitro fertilization (IVF) patients who have micromanipulated embryos replaced. The present study was conducted, in a prospective design, to evaluate effects of corticosteroid on the pregnancy rate in IVF/embryo transfer patients who have non-micromanipulated embryos replaced. Infertile women < 40 years old with tubal factor were included in this study. Patients were grouped according to the different follicle stimulation protocols, and received various doses of 16 beta-methylprednisolone (0, 16 or 60 mg/day) for 4 days from the day of oocyte retrieval. The mean age, duration of infertility, length of folliculogenesis and serum oestradiol concentrations at the time of human chorionic gonadotrophin (HCG) injection were not significantly different between control and corticosteroid-treated groups of patients. Short-term immunosuppression by 16 beta-methylprednisolone administration did not show any effects on the pregnancy and miscarriage rates in IVF/embryo transfer patients. Also, immunosuppression showed no dose effects in any groups. There was no relationship between the types of follicle stimulation protocols and the effect of 16 beta-methylprednisolone. Therefore, we concluded that short-term immunosuppression by exogenous corticosteroids in IVF/embryo transfer patients who have embryos with intact zona pellucida replaced has neither positive nor negative effects on pregnancy rates.

Short-Term Immunosuppression Enhances the Survival of Intracerebral Grafts of A7-Immortalized Glial Cells

The A7 cell line is an astrocyte cell line produced by immortalizing optic nerve astrocytes from the embryonic Sprague-Dawley rat with SV40 large T antigen. In a previous study, we found that grafts of A7 cells into Sprague-Dawley rats survived unreliably and only for a short time after grafting into the striatum. The current experiment was designed to investigate whether short-term immunosuppression with cyclosporin A could enhance the survival of the A7 cells grafted into the adult rat striatum. A7 cells were labeled with 1,1′-dioctadecyl-3,3,3′,3′ -tetramethylindocarbocyanine (DiI) and stereotactically grafted into the left striatum of adult rat brains. Some of the host animals received cyclosporin A until 30 or 60 days after grafting. Animals were sacrificed after periods of up to 90 days, and the number and location of surviving A7 cells were determined by counting the DiI-labeled A7 cells. The possible infiltration of the host animal immune cells into the A7 grafts was evaluated using antibodies that recognize lymphocyte and macrophage antigens (anti-CD8 and anti-CD4). There was a dramatic loss of cells with time in the untreated control animals. Short-term immunosuppression with cyclosporin A significantly increased the average number of surviving A7 cells at all time points examined (30, 60, and 90 days). There was no difference in the number of surviving A7 cells between animals that received a short course of immunosuppression and those that received continuous immunosuppression for 60 days. In general, the levels of CD4 immunoreactivity in the vicinity of the A7 grafts were inversely correlated with A7 cell survival. This suggests that the host animals reacted to the A7 grafts as allografts. These results indicate that the short-term immunosuppression regimen is capable of enhancing the survival of intrastriatal grafts of the A7 astrocyte cell line.

Cell migration and chimerism after whole-organ transplantation: The basis of graft acceptance

Improvements in the prevention or control of rejection of the kidney and liver have been largely interchangeable (1, 2) and then applicable, with very little modification, to thoracic and other organs. However, the mechanism by which anti rejection treatment permits any of these grafts to be “accepted” has been an immunological enigma (3, 4). We have proposed recently that the exchange of migratory leukocytes between the transplant and the recipient with consequent long-term cellular chimerism in both is the basis for acceptance of all whole-organ allografts and xenografts (5). Although such chimerism was demonstrated only a few months ago, the observations have increased our insight into transplantation immunology and have encouraged the development of alternative therapeutic strategies (6).