Primary immunosuppression with mycophenolate mofetil and antithymocyte globulin for kidney transplant recipients of a suboptimal graft

BACKGROUND: In renal transplantation the beneficial immunosuppressive effects of cyclosporin (CsA) may be curtailed by its nephrotoxicity, specially in patients receiving a cadaveric allograft from suboptimal donors or at risk of delayed graft function. Mycophenolate mofetil (MMF) and antithymocyte globulin (ATG) have each demonstrated to be potent immunosuppressants in renal transplantation. In a prospective analysis we have studied the results at 6 months of the combination of MMF, ATG and low-dose steroids in patients with low immunological risk receiving a first cadaveric renal allograft from a suboptimal donor or at risk of delayed graft function. METHODS: Patients with preformed reactive antibodies < 500% receiving a first graft from a suboptimal donor (age > or = 40 years, non-heart-beating, acute renal failure, arterial hypertension) or at risk of delayed graft function (cold ischaemia time > or = 24 h) were eligible for this open single-arm pilot trial. From September 1996 to March 1997 we recruited 17 patients. They were treated with MMF 2 g p.o. preoperatively, and after transplantation at 3 g/day; rabbit ATG i.v. at 2 mg/kg preoperatively, and 1.5 mg/kg/day the first day after transplantation, followed by four doses of 1 mg/kg on alternate days; prednisone was given at 0.25 mg/kg/day and reduced progressively to 0.1 mg/kg/day at 3 months. Primary outcomes were incidence of biopsy-proven acute rejection, delayed graft function, opportunistic infections, graft and patient survival, and the need for introduction of CsA treatment. RESULTS: delayed graft function occurred in two cases (12%). Four of 17 patients (24%) had a biopsy-proven acute rejection (2 grade I and 2 grade II) within the first 3 months after transplantation. CsA was added in two cases with grade II biopsy-proven acute rejection, and in one with grade I biopsy-proven acute rejection. In one patient MMF was replaced by CsA because of gastrointestinal intolerance. Mean serum creatinine 6 months after transplantation was 159+/-59 micromol/1. Cytomegalovirus tissue invasive disease occurred in one patient (6%). At 6 months follow-up all patients are alive with functioning allografts. CONCLUSIONS: These preliminary results suggest that in low-immunological-risk patients who receive a suboptimal renal allograft or at risk of delayed graft function, the combination of MMF, ATG, and steroids is an efficient immunosuppressive regime that may avoid the use of CsA in 70% of the recipients.     

Anatomy of schizophrenia revisited

BACKGROUND: The purpose of this study was to investigate whether obliterative bronchiolitis might occur after xenogenic pulmonary transplantation. A model for obliterative airway disease (OAD) after tracheal allograft transplantation in the rat undergoes tracheal obliteration with histologic features characteristic of obliterative bronchiolitis in human lung transplant recipients. Using this model, the pathogenesis of OAD and its prevention with immunosuppressive drugs was studied in rat recipients of hamster tracheal grafts. METHODS: Tracheae from 30 hamsters (xenografts) or 23 Brown-Norway rats (allografts) were implanted and wrapped in the greater omentum of untreated Lewis rats. The grafts were removed on day 1, 3, 7, 14, 21, or 28 after transplantation and stained with hematoxylin and eosin and Masson's trichrome and by immunohistochemistry and immunofluorescence (IFL) techniques. In addition, 25 recipients were treated with cyclosporine (CsA, 10 mg/kg p.o.), leflunomide (LFM, 20 mg/kg p.o.), or rapamycin (RPM, 6 mg/kg i.p.) for 14 or 21 days (5 animals per treatment group). Visual and morphometric analyses were used to evaluate the extent of airway obliteration, luminal coverage by respiratory or flattened cuboidal epithelium, and extent and density of peritracheal cellular inflammation. RESULTS: In all xenografts, a neutrophilic infiltration of the mucosa and submucosa was observed from day 1 until day 14 and was associated with complete loss of tracheal epithelium by day 14. A marked peritracheal mononuclear cellular infiltrate mixed with plasma cells and eosinophils was seen on days 7 and 14. Both the extent of peritracheal inflammation and the density of the mononuclear cell infiltrate were significantly increased in xenograft tracheae when compared with the allografts. Tracheal obliteration began on day 14 and reached a maximum of 43% on day 21 with evidence of intraluminal fibrosis. In contrast to IFL of allografts, IFL of xenografts demonstrated marked deposition of rat immunoglobulin in the peritracheal tissue on days 7 and 14. The effects of treatment with immunosuppressive drugs on tracheal graft narrowing and protection of respiratory epithelium were as follows: After 14 days of treatment, the percentage of tracheal graft narrowing was 12%, 23%, and 19% in the no treatment, CsA, and LFM groups, respectively; the percentage of respiratory epithelium at 14 days was 0%, 21%, and 95%. After 21 days of treatment, the percentage of tracheal graft narrowing was 43%, 49%, 12%, and 5% for the no treatment, CsA, LFM, and RPM groups, respectively; the percentage of respiratory epithelium at 21 days was 0%, 39%, 86%, and 0%. Using computerized morphometry, the extent and densities of the peritracheal cellular infiltrates were significantly reduced in LFM- and CsA-treated groups when compared with untreated xenograft controls. LFM and RPM, but not CsA, significantly reduced the degree of luminal obliteration compared with no treatment (P<0.05). LFM and, to a lesser extent, CsA were able to prevent the loss of normal respiratory epithelium. Analysis by IFL revealed a marked decrease in rat immunoglobulin deposition in xenografts from LFM- and RPM-treated groups compared with xenografts from CsA-treated or untreated rats. CONCLUSIONS: (1) OAD occurs not only after tracheal allotransplantation but also after xenotransplantation. (2) Subepithelial infiltration of neutrophils and the appearance of plasma cells and eosinophils in the peritracheal infiltrates distinguished the histology of rejected xenografts from allografts. (3) Antibody deposition was detected by IFL only in xenografts. (4) Treatment with LFM or RPM significantly decreased the severity of luminal obliteration. Importantly, LFM also prevented the loss of respiratory epithelium.     

Mycophenolate mofetil. A useful immunosuppressive in inflammatory eye disease

OBJECTIVE: To assess the usefulness of mycophenolate mofetil (MMF) (Cellcept, Roche), a potent selective uncompetitive and reversible inhibitor of ionisine monophosphate dehydrogenase involved in purine synthesis, as an immunosuppressive and steroid-sparing agent in the management of ocular inflammatory disease. DESIGN: Open-label, prospective, uncontrolled pilot study. PARTICIPANTS: Eleven patients with uncontrolled ocular inflammation. INTERVENTION: Mycophenolate mofetil, at a dosage of 1 g twice daily, was given in conjunction with steroids, as a steroid-sparing agent, or as an additional agent with cyclosporine (CsA), or instead of CsA or azathioprine. MAIN OUTCOME MEASURES: The inflammatory response, side effects, and toxicity were monitored. RESULTS: The addition of MMF to immunosuppressive regimens led to the improvement in symptoms and the ability to reduce the dose of prednisone in most patients. Ten of 11 patients showed a favorable response to MMF, with few side effects noted. CONCLUSION: These findings suggest that MMF is a useful immunosuppressive drug for controlling ocular inflammation with minimal side effects.     

Localization of Six4/AREC3 in the developing mouse retina; implications in mammalian retinal development

BACKGROUND: We designed an antisense phosphorothioate oligodeoxynucleotide (oligo) to specifically inhibit the expression of rat intercellular adhesion molecule-1 (ICAM-1) mRNA (IP-9125). METHODS: IP-9125 oligo was delivered intravenously by osmotic pump alone or in combination with cyclosporine (CsA) to recipients in order to prevent the rejection of kidney or heart allografts. In additional experiments, kidney allografts were perfused with IP-9125 before grafting. RESULTS: IP-9125 inhibited ICAM-1 mRNA and ICAM-1 protein expression in rat aortic endothelial cells; scrambled controls IP-12140 and IP-13944 were ineffective. Untreated ACI (RT1a) recipients rejected Lewis (RT1l) kidney allografts at a mean survival time of 8.5+/-1.1 days. A 14-day intravenous administration of 2.5 mg/kg/day IP-9125 prolonged the survival of kidney allografts to 39.2+/-16.4 days; 5.0 mg/kg/day, to 43.0+/-17.5 days; and 10.0 mg/kg/day, to 50.4+/-21.6 days. In contrast, a scrambled control IP-12140 was not effective. A combination of 10 mg/kg/day IP-9125 and 1.0 mg/kg/day CsA delivered for 14 days synergistically extended kidney allograft survival times 88.5+/-7.5 days. In contrast, the combination of 10.0 mg/kg/day control IP-12140 with CsA was ineffective (20.7+/-3.2 days) when compared with CsA alone (20.2+/-4.0 days). Similar results were obtained for heart transplants in recipients treated with IP-9125 alone or in combination with CsA. Furthermore, in situ immunostaining showed that IP-9125 significantly reduced the expression of ICAM-1 protein in kidney allografts. Finally, perfusion of kidney grafts alone with 20.0 mg per 2 ml of IP-9125 protected kidney allografts from rejection (37.5+/-7.5 days; P < 0.001), whereas perfusion with 20 mg per 2 ml of control IP-12140 was ineffective (12.6+/-5.0 days). CONCLUSIONS: Rat ICAM-1 IP-9125 oligo inhibits ICAM-1 protein expression in vitro and in vivo as well as blocks allograft rejection when used for pretreatment of donors, graft perfusion, or postoperative treatment of recipients.     

Mycophenolate mofetil, together with cyclosporin A, prevents anti-OKT3 antibody response in kidney transplant recipients

OKT3 monoclonal antibody, a murine IgG2a monoclonal antibody targeting the T cell CD3 antigen, elicits a neutralizing humoral response in 20 to 50% of kidney transplant recipients when the concomitant immunosuppression consists of CsA-Sandimmun (SAND) and azathioprine (AZA). In the present study, we investigated the impact of the newer agents, CsA-Neoral (NEO) and mycophenolate mofetil (MMF) on OKT3 sensitization. Sixty-two consecutive kidney transplant recipients received prophylactic OKT3 (5 mg/d) from days 0 to 13, together with steroids. Concomitant immunosuppression consisted of either AZA + SAND (n=20), AZA + NEO (n=31), or MMF + NEO (n=11). The following doses were used: AZA, 2 mg/kg per d from days 0 to 13, then 1 mg/kg per d; MMF, 2 g/d starting on day 1; and CsA, either SAND or NEO, 6 mg/kg per d from day 6. At least two serum samples per month were available during the initial 3 mo for each patient. IgG anti-OKT3 antibodies were first evaluated by enzyme-linked immunosorbent assay. Patients were considered sensitized if their serum scored positive at a dilution > or = 1/1000. Peak titers of IgG anti-OKT3 antibodies and the incidence of patients harboring neutralizing anti-idiotypic antibodies were also determined. A first reduction in OKT3 sensitization was seen in patients receiving Neoral instead of Sandimmun (AZA + SAND: 10 of 20 [50%] patients sensitized versus 6 of 31 [19%] in the AZA + NEO group; P=0.03). This was probably related to the achievement of higher mean CsA trough blood levels in the NEO group during the first month (253+/-44 versus 186+/-49 ng/ml in SAND patients). Peak antibody titers and the proportion of patients with anti-idiotypic antibodies were similar in the AZA + SAND and AZA + NEO groups. A further reduction in the sensitization rate was observed with the replacement of AZA by MMF (MMF + NEO: 0% sensitized patients; P=0.0013). It is concluded that the combination of CsA-Neoral and MMF efficiently prevents sensitization against OKT3.     

Cyclosporine A inhibits lymphocyte migration into ovine peripheral nerve allografts

Lymphocyte migration into nerve allografts was measured to estimate the cyclosporine A (CsA) dose required to suppress rejection. Twelve outbred sheep received daily subcutaneous CsA at 0, 5, 10, or 15 mg/kg/day for 2 weeks prior to implantation of multiple heterotopic subcutaneous nerve grafts. Lymphocyte migration was determined after 7 days by an intravenous pulse of autologous 111indium-labeled lymphocytes and subsequent quantitation of gamma radioactivity in nerve tissue (CPM/g, mean +/- SEM). Measurement by radioimmunoassay revealed a dose-dependent increase in blood cyclosporine levels. Lymphocyte migration into autografts (404+/-44) was significantly less than migration into allografts (16,554+/-2,049), in control animals (P < 0.01). A dose-dependent inhibition of lymphocyte migration into nerve allografts was observed with counts of 7,662+/-1,692, 4,083+/-1,112, and 1,561+/-232 in sheep receiving 5, 10, or 15 mg/kg/day of CsA, respectively. Daily CsA administration produced effective blood levels and immunosuppression sufficient to inhibit lymphocyte migration into nerve allografts.     

A comparison of the antigen-presenting capabilities of class II MHC-expressing human lung epithelial and endothelial cells

Human lung alveolar epithelial cells constitutively express class II major histocompatibility complex (MHC). Human lung microvascular endothelial and small airway epithelial cells can be induced to express class II MHC by stimulation with the pro-inflammatory cytokine interferon-gamma. The levels of class II MHC on lung epithelial and endothelial cells were comparable to those seen on an Epstein-Barr virus (EBV)-transformed B-cell line. However, the costimulatory molecules B7-1 and B7-2 were not expressed. The ability of the class II MHC expressing human lung parenchymal cells to present alloantigen to CD4+ T lymphocytes was investigated. Freshly isolated human alveolar epithelial cells (type II pneumocytes) and monolayers of interferon-gamma-stimulated small airway epithelial and lung microvascular endothelial cells were co-cultured with allogeneic CD4+ T lymphocytes and proliferation determined by [3H]thymidine incorporation. A clear difference was observed between effects of the epithelial and endothelial cells on CD4+ T-lymphocyte activation. Alveolar and small airway epithelial cells failed to stimulate the proliferation of allogeneic CD4+ T lymphocytes whereas lung microvascular endothelial cells did stimulate proliferation. This difference could not be explained by the levels of class II MHC or the lack of B7-1 and B7-2 solely. Microvascular endothelial cells, and not alveolar or small airway epithelial cells, possess B7-independent costimulatory pathways.     

Estradiol inhibits allograft-inducible major histocompatibility complex class II antigen expression and transplant arteriosclerosis in the absence of immunosuppression

BACKGROUND: The etiology of transplant arteriosclerosis is unknown, but current data point to the alloimmune response. Previously, we found that estradiol-17beta (E2) with immunosuppressant cyclosporine abolishes major histocompatibility complex (MHC) class II expression in the allograft. This study determines the effect of E2 on MHC class II antigen expression in the allograft, in the absence of immunosuppression. METHODS: Lewis male rats received orthotopic abdominal aorta allografts from male Brown-Norway rats. The recipients were treated continuously subcutaneously with either 20 microg x kg(-1) x day1 of E2 (n=20) or placebo (n=20), from 2 days before transplantation until death on posttransplant days 1, 3, 7, and 14. The allografts were harvested and processed for morphometry and for immunohistochemical staining of MHC class II antigens, macrophages, CD4 and CD8 T lymphocytes, interferon-gamma (IFN-gamma), and IFN-gamma receptor. RESULTS: With E2 treatment, we observed that inducible MHC class II antigen expression is abolished in the media of the vascular allograft; the expression of IFN-gamma and IFN-gamma receptor is unaffected; and macrophage infiltration of the vascular allograft is inhibited significantly (P<0.01), whereas the CD4 and CD8 T lymphocytes are not significantly (P=0.07) suppressed. The myointimal hyperplasia in the allografts from E2-treated-recipients was 3-4-fold less than that from the placebo-treated recipients. CONCLUSIONS: Without immunosuppression, E2 inhibition of transplant arteriosclerosis is still associated with inhibition of inducible MHC class II antigen expression in the allografts. The estradiol-17beta abolition of inducible MHC class II antigen expression in the aorta allograft occurs in spite of up-regulation of IFN-gamma ligand and receptor protein.     

Prophylaxis of acute renal allograft rejection using FTY720 in combination with subtherapeutic doses of cyclosporine

BACKGROUND: In rodent transplant models, FTY720 exerts a synergistic affect with cyclosporine (CsA) to prolong allograft survival. The present experiments sought to test this combination in subhuman primates. METHODS: Cynomolgus monkeys were transplanted with kidney allografts that were incompatible in mixed lymphocyte culture reactions. The animals were treated with daily intramuscular injections of CsA using doses selected to maintain whole blood trough concentrations at therapeutic values between 40 and 200 ng/ml. The 4 experimental groups included CsA without or with 0.1, 0.3, or 1 mg/kg/day FTY720 delivered daily by intravenous bolus injection. Therapeutic effects were suggested both by the graft histology of biopsy within the first 10 posttransplant days and by the length of host survival. RESULTS: Whereas recipients treated with CsA alone rejected kidney allografts at a median survival time of 8.5 days (n=4), those treated with either 0.1 or 0.3 mg/kg/day FTY720 in addition to CsA showed significant prolongation of kidney allograft survival to 71 days (n=3; P<0.04) or 63 days (n=5; P<0.05), respectively. The hosts in the 1.0 mg/kg/day FTY720 group survived 48 days, with 2 of 5 recipients succumbing at 9 or 17 days postgraft, suggesting possible complications caused by overimmunosuppression. Biopsies of the 0.1 mg/kg/day FTY720 group on posttransplant day 7 documented mild to moderate rejection (grade I), indicated by multiple focal areas of tubular destruction. The histology results of transplants in the 0.3 or 1 mg/kg/day FTY720 group showed only minimal interstitial inflammatory infiltrates (borderline grade), with no evidence of tubular or arterial damage. Serum creatinine values among the animals in the 0.1 mg/kg/day FTY720 group showed increases in 2 of 3 recipients by day 20 and in the third by day 41 postgraft. Among the 0.3 mg/kg/day FTY720 group, 3 of 5 recipients maintained baseline creatinine values to 45 days postgraft; 1 recipient had stable kidney function for 120 days postgraft. CONCLUSIONS: Addition of FTY720 therapy to a subtherapeutic CsA immunosuppressive regimen delays the rejection of renal allografts in subhuman primates.     

Selective training of the vastus medialis muscle using electrical stimulator for chondromalacia patella

Islet allografts transplanted into Type I diabetic recipients may be destroyed by allorejection or recurrent autoimmune diabetes. We studied islet transplantation in three murine models in order to determine the relative sensitivity of autoimmunity and alloimmunity to two immunosuppressive agents that may be useful in clinical islet transplantation: 15-deoxyspergualin (DSG) and anti-CD4 antibody (GK 1.5). In the model in which only allorejection occurs (BALB/c islets transplanted into streptozotocin-induced diabetic CBA or streptozotocin-induced diabetic NOD recipients), both DSG and anti-CD4 antibody treatment led to indefinite survival of allogeneic islets (>100 days in both treatments). In the second model in which only recurrent autoimmunity can destroy islet grafts (islets from NOD donors transplanted into spontaneously diabetic NOD recipients), only anti-CD4 treatment caused prolonged graft survival [MST 36.7 +/- 6.8 days vs 9.8 +/- 1.8 days (controls), P < 0.0002]. Treatment with DSG did not cause any increase in graft survival (MST 12.6 +/- 5.4 days, NS). Finally, using a model in which both autoimmunity and allorejection may occur (BALB/c to spontaneously diabetic NOD mice), treatment with anti-CD4 caused marked graft prolongation [42.0 +/- 14.5 days vs 7.2 +/- 0.8 days (control), P < 0.002] while DSG again did not prolong graft survival with respect to untreated recipients (9.8 +/- 3.0, NS). We conclude that recurrent autoimmunity in the NOD mouse involves a CD4+ T cell that is not sensitive to DSG. Anti-CD4 antibody may be useful in human clinical islet transplantation trials because it seems to prevent both allorejection and recurrent autoimmunity.     

Experimental study of tracheal allotransplantation with cryopreserved grafts

OBJECTIVE: Tracheal reconstruction is necessary in patients with extensive tracheal stenosis caused by neoplasm, trauma, and congenital disease. We investigated the possibility of tracheal allotransplantation with cryopreserved grafts in a canine model. METHODS: A seven-ring section of thoracic trachea was removed in 19 adult mongrel dogs. In group A (n = 4), a five-ring tracheal autograft was implanted. In group B (n = 6), a five-ring allograft was implanted without immunosuppression. In group C (n = 9), a five-ring cryopreserved tracheal allograft was implanted without immunosuppression. Omentopexy wrapping around the grafts and both anastomotic sites was used in all animals. RESULTS: All grafts survived without any evidence of atrophy or stenosis in group A. All animals in group B died of severe airway obstruction within 1 month, and postmortem examination of these grafts showed epithelial defect and necrotic tracheal cartilage in the scar tissue. In group C, no animals died of asphyxia caused by severe stenosis of the grafts. The graft epithelium was no longer present 20 days after transplantation, and the graft was covered with regenerated epithelium within about 60 days after the operation. CONCLUSION: These findings show that cryopreserved tracheal allografts can be transplanted by means of omentopexy without immunosuppression and that cryopreservation may reduce tracheal allogenicity.     

Oral administration of rapamycin and cyclosporine differentially alter intestinal function in rabbits

The immunosuppressive drugs rapamycin (Rap) and cyclosporine A (CsA) are used clinically to modify or abolish immune-mediated functions. This study examined the effect of orally administered regimens of Rap, CsA, and a combination of Rap/CsA on intestinal function in male New Zealand white rabbits. Animals received oral doses of CsA (15 mg/kg/body weight/day), low-dose (LD) and high-dose (HD) Rap (0.25 or 1 mg/kg/body wt/day, respectively), or Rap/CsA (0.25 and 5 mg/kg/body wt/day, or 0.5 and 5 mg/kg/body wt/day, respectively) for 20 days. We measured in vitro uptake of nutrients and permeability, and morphometric measurements in the jejunum and ileum were made. Animals receiving HD-Rap or HD-Rap/CsA had decreased food intake, body weight, and intestinal weight, when compared with LD-Rap, LD-Rap/CsA, CsA, or controls. The maximal transport rate (Vmax) for the active jejunal uptake of D-glucose was increased in HD-Rap and CsA, but not in the HD-Rap/CsA-treated animals. The jejunal Vmax of D-glucose in the LD-Rap- or -Rap/CsA-treated animals was no different from controls. In the HD-Rap- and HD-Rap/ CsA-treated animals, jejunal rates of uptake of stearic, linoleic, and linolenic acids were reduced when compared with controls. Jejunal and ileal permeability (as assessed by the passive uptake of L-glucose, tissue conductance, and mucosal-to-serosal flux of [3H]inulin) was increased in animals treated with HD-Rap or HD-Rap/CsA, when compared with CsA or controls. These parameters of permeability were no different at lower doses of Rap or Rap/CsA. The jejunal and ileal villous surface area was increased in CsA, but decreased in HD-Rap or HD-Rap/CsA animals. Thus, HD-Rap given alone or in combination with CsA reduced body weight gain, in part due to reduced food intake and malabsorption of lipids, which was due at least in part to reduced intestinal surface area. The relevance of these findings to patients undergoing chronic immunosuppressive drug therapy needs to be established.     

Phase I trial of intravenous carboplatin and cyclosporin A in refractory gynecologic cancer patients

Our objective was to determine the maximum tolerated dose of cyclosporin A (CsA) delivered as a loading dose (LD) and continuous i.v. infusion (CI) in combination with carboplatin in patients with refractory gynecologic cancers. Twenty-nine heavily pretreated patients (25 ovarian epithelial, 2 cervical, and 2 endometrial carcinomas) received 113 cycles of CsA and carboplatin from September 1989 to September 1991. Twenty-four of these 29 carcinomas were strictly defined to be platinum resistant. CsA was administered as a LD escalated from 6 to 10 mg/kg followed by a 24-h CI from 2.5 to 14.5 mg/kg/day. Carboplatin was targeted to an area under the time versus concentration curve (AUC) of 6 mg/ml x min and was not dose escalated. Whole-blood CsA concentrations (fluorescence polarization immunoassay) at the maximum tolerated dose (10 mg/kg LD, 14.5 mg/kg/day CI) ranged from 2.4 to 3.0 microgram/ml over 12 h. Estimated median carboplatin AUC, based on calculated carboplatin clearance, was 7.9 mg/ml x min. The dose-limiting toxicity of the combination of CsA and carboplatin was grade 4 thrombocytopenia. Grade 3 or 4 thrombocytopenia occurred in 35% of the patients, which could be explained by the effects of carboplatin (AUC of 6 mg/ml x min) alone. Overall, neutropenia occurred in 24% of the patients and anemia in 17% of the patients. Grade 3 or 4 nausea or vomiting was noted in 10 and 14% of the patients, respectively. Grade 3 hypertension during CsA administration occurred in 14% of the patients. No grade 3 or 4 nephrotoxicity was seen in this trial. Three objective responses were noted: one complete response (11 months) and one partial response (5 months), both in potentially platinum-sensitive patients with platinum-free intervals of only 9 months each. One platinum-resistant patient had a partial response for 21 months. Five additional patients experienced >75% reduction of CA-125 or a return to a normal CA-125 titer. We concluded that whole-blood CsA concentrations of >3.0 microgram/ml (as seen when CsA is used as a modulator of multidrug resistance) were not achievable in this combination with carboplatin in this population of heavily pretreated gynecologic cancer patients. However, because CsA is used in this trial as a chemosensitizer in platinum-sensitive tumors and as a chemomodulator of platinum resistance, we targeted a CsA concentration of >1.0 microgram/ml, which was achieved. The CsA dose recommended for a Phase II trial of this combination is 10 mg/kg LD and 11.6 mg/kg/day CI, which results in blood CsA concentrations ranging from 1.2 to 1.3 microgram/ml over 12 h. Responses in this population of refractory gynecologic cancer patients are unusual, and these encouraging results form the basis for a Phase II trial of this combination.     

Intermittent administration of 15-deoxyspergualin for acute on chronic rejection after renal transplantation

We used 15-deoxyspergualin (DSG) to treat acute on chronic rejection (AOCR) in six kidney transplant recipients. DSG was administered intermittently at a dose of 200-300 mg per body every 2 or 4 weeks for more than 6 months. The efficacy of DSG was evaluated by the changes in serum creatinine values during the rejection therapy. Four (67%) of the six patients responded with the suppression of the increase in serum creatinine values. On the other hand, one patient did not respond at all and developed advanced pancytopenia. These findings suggested that intermittent administration of DSG would be efficient therapy on AOCR in the renal allografts if the patients were treated carefully to prevent severe side effects.     

Pedicle instrumentation in the thoracic spine. A morphometric and cadaveric study for placement of screws

The aim of this study was to examine the possible effects of local application of a potent immunosuppressive drug, cyclosporin A (CsA), on fibroblast proliferation in vitro. Rabbit subconjunctival fibroblasts were cultured and incubated with varying doses of CsA (from 0.5 to 5%). Growth inhibition curves were obtained, and cell attachment and reversibility of drug-induced growth inhibition were studied. None of the doses stimulated fibroblast proliferation. CsA inhibited fibroblast proliferation depending on the dose and duration of application. Also, drug-induced growth inhibition decreased depending on dose and duration of application. While the dosage which led to 50% inhibition was found to be 7.2 mg/ml, 4% and higher doses had toxic effects.     

Administration-time-dependent effects of diltiazem on the 24-hour blood pressure profile of essential hypertension patients

Cells of the central nervous system (CNS) normally do not express detectable levels of major histocompatibility complex (MHC) Class I antigens. However, MHC Class I expression can be induced after virus infection. We tested the hypothesis that virus-induced Class I expression is mediated by lymphocytes or cytokines using lymphocyte- and cytokine-deficient mice. We used Theiler's murine encephalomyelitis virus (TMEV), which induces CNS demyelination that maps genetically to the D region of MHC Class I and is associated with high levels of Class I products. TMEV infection of severe combined immunodeficiency (SCID) and recombination activation gene-1-deficient mice, which lack B and T lymphocytes, resulted in equivalent H-2D and H-2K expression in brain and spinal cord, according to analysis of the area and intensity of immunoperoxidase staining. Class I antigens were demonstrated as early as 6 hours after infection, and they were more widely distributed than viral RNA, indicating that expression was induced indirectly via a soluble factor. To determine whether cytokines induced the expression, we infected mice lacking receptors for interferon-alpha/beta (IFN-alpha/beta R (-/-)), interferon-gamma (IFN-gamma R(-/-)), and tumor necrosis factor-alpha (TNFRp55(-/-)). TMEV-infected IFN-gamma R(-/-) and TN-FRp55(-/-) mice expressed Class I antigens in the CNS, whereas IFN-alpha/beta R(-/-) mice did not, establishing that IFN-alpha/beta mediated the expression. In contrast to the equivalent expression in SCID mice, we observed greater area and higher intensity of H-2D versus H-2K antigens in infected SCID mice reconstituted with normal spleen cells. Collectively, the data indicate that after TMEV infection, early generalized MHC Class I expression is mediated by IFN-alpha/beta independently of lymphocytes, but the differential regulation of H-2D over H-2K may be controlled by B and/or T lymphocytes.     

Induction of MHC-class I restricted human suppressor T cells by peptide priming in vitro

The induction of regulatory T cells may offer an effective means for specific immunosuppression of autoimmune disease and allograft rejection. The existence of suppressor T cells has been previously documented, yet their mechanism of action remains poorly characterized. Our studies demonstrate that T suppressor (Ts) cell lines can be generated by in vitro immunization of human PBMCs, with synthetic peptides or soluble proteins coupled to beads. Such Ts cells express the CD8+CD28- phenotype and show the following characteristics: (a) antigen specificity and restriction by self MHC Class I molecules; (b) limited TCR V beta gene usage; (c) ability to inhibit antigen-specific, MHC Class II restricted, Th proliferative responses; and (d) capacity to downregulate and/or inhibit the upregulation by Th of CD40, CD80, and CD86 molecules on APCs. The inhibitory activity of Ts on Th proliferation requires the tripartite interaction between Th, Ts, and APCs and results from inefficient costimulation of Th.     

FK 506 'rescue' immunosuppression for obliterative bronchiolitis after lung transplantation

PRELIMINARY EXPERIENCE: In a consecutive case series (level V evidence) involving 10 recipients of unilateral lung transplantation (LT) with bronchiolitis obliterans, in conjunction with Fujisawa protocol 93-0-003, the physiologic responses to FK 506 (tacrolimus) "rescue" immunosuppression were assessed. Recipients were 22+/-18 months post-LT and demonstrated progressive allograft dysfunction that was refractory to pulsed-dose methylprednisolone therapy. All recipients received induction immunosuppression with Minnesota antilymphocyte globulin (10 to 15 mg/kg/d) for 5 to 10 days, cyclosporine (CsA) (whole-blood Abbott TDX fluorescence polarization immunoassay (Abbott Inc, Abbott Park, IL)=600 to 800 ng/mL), azathioprine (2 mg/kg/d), and prednisone (tapered to 0.2 mg/kg/d). The "rescue" regimen consisted of oral FK 506 adjusted to maintain a whole-blood Abbott IMX microparticle enzyme immunoassay (Abbott Inc, Abbott Park, IL) of 10 to 15 ng/mL with an initial increase in prednisone (1.0 mg/kg/d) during conversion that was subsequently tapered to 0.2 mg/kg/d. Spirometry was performed monthly in accordance with accepted American Thoracic Society criteria. Recipients were classified in accordance with the International Society for Heart and Lung Transplantation (ISHLT) "Working Formulation for Standardization of Nomenclature and for Clinical Staging of Chronic Dysfunction in Lung Allografts" as stages Ib (n=2), IIb (n=4), and IIIb (n=4) upon entry to the protocol. The deltaFEV1/month relationships during CsA- and FK 506-based regimens were analyzed by linear regression and compared by signed rank test (p<0.05). RESULTS: The deltaFEV1/month slopes were -0.0687+/-0.0221 and +0.0300+/-0.033 L/mo (mean+/-SEM) for CsA and FK 506, respectively (p=0.037). Although no significant spirometric improvement was noted in most recipients, no further decline in FEV1 occurred after conversion to FK 506. Recipients with less severe chronic dysfunction (ie, obliterative bronchiolitis [OB] stages Ib and IIb) stabilized their spirometric indexes at higher levels. Two recipients with OB stage IIIb died of hypercapnic respiratory failure at 6 and 8 months after conversion. CONCLUSIONS: The deltaFEV1/mo slopes stabilized after FK 506 conversion. Earlier conversion may be beneficial in stabilizing FEV1 at a higher plateau. Significant economic impact may be anticipated with FK 506 compared to alternative cytolytic strategies for OB. However, multicenter prospective controlled investigations are necessary to further address the potential role of FK 506 after LT (level I evidence). Furthermore, the ISHLT "Staging of OB Syndrome" may have significant clinical implications vis-à-vis prognosis and potential therapies.     

Evaluation of the Biolog system for the identification of food and beverage yeasts

OBJECTIVE: The aim of this study was to examine the relationship between erythrocyte-to-plasma distribution ratio of cyclosporin (CsA-EP) and lymphocyte proliferation as an indicator of immunosuppressive activity in renal transplant patients. METHODS: A total of 113 whole blood samples obtained from 6 inpatients with renal transplantation were analysed. CsA concentrations in blood and plasma at trough were measured by fluorescence polarization immunoassay using monoclonal antibody, lymphocyte proliferation in response to phytohaemagglutinin was evaluated by the fluorimetric derivatization method using ethidium bromide and the stimulation index (SI) was calculated. RESULTS: There was no correlation between CsA dose and trough levels (vs blood CsA, r2 = 0.052; vs plasma CsA, r2 = 0.054, n = 113). A significant negative correlation between the SI and the CsA-EP was found in individual or all samples (r2 = 0.224, p < 0.0001, n = 113), whereas CsA trough levels in blood or plasma had no correlation with the SI. CONCLUSION: Although the degree of contribution of CsA-EP to the SI was 22%, the CsA-EP is a more useful predictor of changes in immunosuppressive response than CsA concentration in blood or plasma. The adoption of the CsA-EP as a monitoring index could be helpful in assessing the appropriateness of CsA immunosuppressive therapy.     

Long-term composite tissue allograft survival in a porcine model with cyclosporine/mycophenolate mofetil therapy

BACKGROUND: Low-dose cyclosporine (CsA)/mycophenolate mofetil (MMF) therapy has significantly reduced the frequency of rejection and drug-induced side effects in rat hindlimb allograft recipients. With an eye toward direct clinical application, we developed a large-animal extremity composite tissue allograft model to assess the antirejection efficacy and systemic toxicity of combination CsA/MMF treatment. METHODS: Radial forelimb osteomyocutaneous flap transplants were performed between size-matched, outbred pigs assigned to one of two groups: 5 control pigs received no immunosuppression, and 10 pigs received a once-daily oral CsA/MMF/prednisone regimen. Rejection was assessed by visual inspection of flap skin and correlated with serial histopathologic examination of skin biopsies. RESULTS: In all control pigs, the flap was completely rejected on day 7. Of the 10 pigs receiving treatment, one died from pneumonia and an another from an anesthetic complication on days 19 and 30, respectively, without signs of rejection. Two flaps were lost on days 25 and 29 from severe rejection. Three pigs were free of rejection at the end of the 90-day follow-up period, and three had stable mild-to-moderate rejection at 90 days (P= 0.0007 vs. controls). White blood cell and platelet counts, serum creatinine values, and liver function tests remained normal in all animals receiving immunosuppressive therapy. CONCLUSIONS: Our results, to our knowledge, demonstrate for the first time that rejection can be significantly delayed in a large-animal composite tissue allograft model including skin using only orally administered agents dosed according to clinically relevant strategies without significant drug-specific systemic side effects.