P. B. Carroll

Human islet isolation and allotransplantation in 22 consecutive cases

This report provides our initial experience in islet isolation and intrahepatic allotransplantation in 21 patients. In group 1, 10 patients underwent combined liver-islet allotransplantation following upper-abdominal exenteration for cancer. In group 2, 4 patients received a combined liver-islet allograft for cirrhosis and diabetes. One patients had plasma C-peptide greater than 3 pM and was therefore excluded from analysis. In group 3, 7 patients received 8 combined cadaveric kidney-islet grafts (one retransplant) for end-stage renal disease secondary to type 1 diabetes mellitus. The islets were separated by a modification of the automated method for human islet isolation and the preparation were infused into the portal vein. Immunosuppression was with FK506 (group 1) plus steroids (groups 2 and 3). Six patients in group 1 did not require insulin treatment for 5 to greater than 16 months. In groups 2 and 3 none of the patients became insulin-independent, although decreased insulin requirement and stabilization of diabetes were observed. Our results indicate that rejection is still a major factor limiting the clinical application of islet transplantation in patients with type 1 diabetes mellitus, although other factors such as steroid treatment may contribute to deteriorate islet engraftment and/or function.

IL-8/IL-8 receptor expression in psoriasis and the response to systemic tacrolimus (FK506) therapy.

Recently, the keratinocyte IL‐8/IL‐8 receptor (IL‐8R) pathway has been implicated in the pathogenesis of psoriasis, and there is evidence that the potent macrolide immune suppressant tacrolimus (formerly FK506) can inhibit this pathway in vitro. In this study, determination of the expression of cytokine mRNAs in lesional skin of patients with active disease by reverse transcriptase polymerase chain reaction revealed transcripts for IL‐1β, tumour necrosis factor‐alpha (TNF‐α), IL‐6, IL‐8, IL‐8R, IL‐10, interferon‐gamma (IFN‐γ), IL‐2R and transforming growth factor‐beta (TGF‐β), but not IL‐2 or IL‐4. IL‐8 was the only cytokine expressed in affected skin of all patients but not in clinically normal skin of healthy subjects. In seven CD4+ T cell clones propagated from the lesional skin of an untreated psoriasis patient, IL‐8 was expressed by the skin‐derived T lymphocytes and not by feeder cells (irradiated autologous blood lymphocytes); IL‐1β, IL‐2, IL‐6 and IL‐10 were also expressed by some or all of the T cell clones, IL‐8 mRNA was not detected in the skin of any patient after the start of systemic tacrolimus therapy; IL‐lβ, IL‐6 and IFN‐γ transcripts were also reduced. By 12 weeks, the mean psoriasis area and severity index (PASI) had decreased from 18·8 to 3·8, a reduction of 80%. In the same post‐treatment biopsies, however, message for IL‐8R persisted. Estimation of circulating IL‐8 levels by enzyme immunoassay showed that all patients with detectable IL‐8 before treatment had decreased levels in response to treatment with tacrolimus; reductions in PASI scores were accompanied by decreases in IL‐8 levels, that varied both in rate and extent. Partial relapse, which in a minority of patients followed the initial period of remission, and was precipitated by drug dose reduction, was accompanied by an increase in circulating IL‐8. These findings add credence to the view that the IL‐8/IL‐8R autocrine/paracrine pathway may be important in the pathogenesis of psoriasis. They further suggest that interference with IL‐8 production and/or that of other key chemokines may be an important mechanism underlying the therapeutic efficacy of tacrolimus, and other agents such as cyclosporin A, with similar molecular actions.

FK 506: a novel immunosuppressant for treatment of autoimmune disease: Rationale and preliminary clinical experience at the University of Pittsburgh

FK 506 (Prograf) is a novel macrolide antibiotic isolated from the soil fungus Streptomyces tsukubaensis [24]. Although it is totally distinct in molecular structure from cyclosporin (CsA) (Sandimmune), a cyclic endecapeptide extracted from the fungus Tolypocladium inflatum (Fig. 1), the two drugs share a remarkably similar, selective inhibitory action on the activation and proliferation of CD4+ T helper (TH) lymphocytes [25, 41, 50, 51, 56]. These cells play an essential, central role both in antigen recognition and as the sources of soluble, hormone-like mediators (cytokines) of the cascade of events leading to the expression of immune reactivity. By inhibiting the activation of CD4+ TH cells, FK 506, like CsA, exerts a wide-range of immunosuppressive activities. It is recognized that both drugs prolong solid-organ allograft survival in experimental animals and in man. FK 506, however, is considerably more powerful as an antilymphocytic agent than CsA, as evidenced by the superior potency of the former drug in inhibiting antigen-driven T cell activation, cytokine production and lymphocyte proliferation in vitro [50]. Moreover, the systemic levels of FK 506 required to induce and maintain immune suppression are approximately 100-fold lower than are the blood levels of CsA to achieve the same effect. The immunosuppressive efficacy of CsA in man (in renal transplant recipients and patients receiving bone marrow transplants) was first reported in 1978; in 1989, the first account of the ability of FK 506 to prevent or reverse organ allograft rejection was published [44]. Data obtained over the last 3 years provide good clinical evidence that FK 506 exhibits a narrower range of side effects than does CsA and that, as compared with CsA, FK 506 has greater steroid-sparing activity [45, 46]. Whilst the potential benefits of FK 506 for the prophylaxis and reversal of organ allograft rejection (in particular liver transplant rejection) are becoming recognized, the value of the drug in the treatment of autoimmune disorders is now also beginning to be assessed. In this article (1) a rationale for the use of FK 506 in autoimmune disease, (2) a description of its molecular action and immunosuppressive activities, (3) a consideration of the biological and pharmacological properties of FK 506, (4) a review of its capacity to inhibit a wide variety of experimental autoimmune disorders, and (5) a report on the early clinical experience with FK 506 in the clinical management of a panoply of autoimmune disease seen at the University of Pittsburgh Medical Center (UPMC) will be presented. Moreover, a brief outline of laboratory investigations utilized to monitor the status of T lymphocytes in these patients and a discussion of the side effects of FK 506 will be presented. Throughout, we shall draw upon comparisons between FK 506 and CsA which have been documented in the literature. Fig. 1 The molecular structure of the immunosuppressive macrolide FK 506 (mol. wt. 822 daltons) and of the less powerful, but similarly acting cyclic endecapeptide cyclosporin A (mol. wt. 1203 daltons) Rationale for the use of FK 506 in autoimmune diseases The role of T cells in autoimmunity The therapeutic use of FK 506 in the treatment of autoimmune disease is based on the premise that all of these disorders are T cell driven [39]. It is, therefore, important to examine the evidence that activated CD4+ TH cells and their cytokine products are important both in the induction and maintenance of various diseases such as psoriasis, uveitis, insulin-dependent type-1 diabetes, chronic active hepatitis-autoimmune (CAH-A), rheumatoid arthritis and multiple sclerosis – diseases that are currently being treated with FK 506. In uveitis [11], type-1 diabetes [6], multiple sclerosis [16] and psoriasis [3] for example, T cells are believed to play an important pathogenic role. Much of the evidence to support this view comes from studies in experimental animal models and from in vitro investigations of the adverse or destructive interactions between T cells, antigen-stimulated cytokines and the target tissue affected by the disease process. In the autoimmune liver diseases [26], CAH-A and primary biliary cirrhosis (PBC), and in rheumatoid arthritis [9], there is abundant evidence for the involvement of T cells in the pathogenesis of each disease and, therefore, a rationale for the use of FK 506 in each exists. In recent years the therapeutic efficacy of CsA in uveitis, psoriasis, PBC, CAH-A, and rheumatoid arthritis has been demonstrated [52]. Moreover, CsA has been shown to alter the natural history of type-1 diabetes [4]. The drug has not, however, made a significant impact upon the clinical management of patients with most of these diseases. In autoimmune diseases such as systemic lupus erythematosus (SLE) or the nephrotic syndrome, the rationale for the use of CsA or FK 506 is less clear. Thus, in SLE, humoral immunity appears to be more important than cellular immunity in the pathogenesis of the disease, and in idiopathic nephrotic syndrome the pathogenic mechanisms responsible for the disease process are far from clear. Nevertheless, in nephrotic syndrome, T cell dysfunction, recruitment of B cells, immunoglobulin deposition within the kidney and a central role for lymphokines have been implicated by various authors [7, 8, 18, 54]. Moreover, CsA has been shown to be very effective in the steroid-sensitive nephrotic syndrome, although less so in steroid-resistant patients [52]. A spectrum of autoimmune disorders is shown in Table 1. The predicted efficacy of FK 506 in their treatment is based on the assumption that the role of T cells in these various disease processes is central, and also on experience in animal models of these diseases with either CsA or FK 506. Account is also taken of clinical experience with CsA in these autoimmune disorders. Table 1 Possible mechanisms of autoimmune diseases and predicted responses to FK 506a Evidence that autoimmune diseases are T cell driven There is a large body of additional experimental data which provides supportive evidence to the thesis that autoimmune diseases are driven by T cells and their cytokine products. In addition to the proven efficacy of CsA or FK 506 in many experimental autoimmune diseases, antibodies directed against CD4+ T cells or against the interleukin 2 receptor (IL-2R; expressed on activated T cells) have been shown to be effective therapeutic agents in these animal models. When stimulated with appropriate antigen or monoclonal antibody, T cell clones derived from lesional tissue or peripheral blood secrete cytokines which effect the pathological changes observed in target tissue (e.g., fibroblasts in scleroderma, keratinocytes in psoriasis or islet cells in type-1 diabetes) that are relevant to the disease process observed in vivo. Such antigen-stimulated T cell clones can induce disease when transferred to healthy recipients (e.g., induction of type-1 diabetes, experimental arthritis or allergic encephalomyelitis). Furthermore, in many experimental models of autoimmunity, it can be shown that neonatal thymectomy has a pronounced beneficial effect in preventing development of the disease. For references and discussion see [40].

Resolution of Severe Pyoderma Gangrenosum in a Patient with Streaking Leukocyte Factor Disease after Treatment with Tacrolimus (FK 506)

Severe, lifelong, unresolving pyoderma gangrenosum occurs in association with recurrent episodes of sterile pyoarthrosis and the presence of a serum factor (called streaking leukocyte factor) [1] responsible for enhancing random migration of purified human neutrophils and mononuclear leukocytes in vitro. Pyoderma gangrenosum is only one feature of this unusual disease. Minor trauma of any sort leads to an excessive accumulation of both mononuclear and polymorphonuclear leukocytes in tissue. This causes subcutaneous induration, sterile abscesses, sterile pyoarthrosis, and extensive areas of skin necrosis similar to those occurring in classical pyoderma gangrenosum, except that the lesions are larger and more confluent. The arthritic lesions are characterized by synovial fluid leukocyte counts greater than 100 000/mm3 and a severe synovitis [1]. We describe a patient with the streaking leukocyte factor syndrome who has been treated successfully with tacrolimus (FK 506, Prograf, Fujisawa Pharmaceutical Co., Osaka, Japan). Case Report The patient was a 31-year-old white man who has had a lifelong history of recurrent and chronic sterile abscesses involving the soft tissues, skin, and joints, which began in infancy and was shown to be associated with streaking leukocyte factor in his serum [1]. Because of the hypertrophic and erosive nature of the arthritic process, the patient had surgical procedures on multiple large joints. His history is summarized in Table 1. The patient was referred to the autoimmune clinic at the University of Pittsburgh Medical Center in April 1991 with a well-established clinical, histopathologic, and serologic diagnosis of this syndrome. Before his referral, he had been treated at Columbia-Presbyterian Medical Center (New York, New York) and the National Institutes of Health with plasmapheresis, high doses of steroids, a wide range of immunosuppressive cytotoxic agents, and thalidomide, but none of these treatments yielded a response. Table 1. Chronology of the Patients Medical Problems Laboratory results included normal counts and types of leukocytes, T cells, and T-cell responses to mitogens; a normal nitroblue tetrazolium test; normal in vitro phagocytosis of yeast by leukocytes as well as normal levels of serum immunoglobulins, complement factors C1q, C4, C5, C1INH, and total hemolytic complement. The laboratory abnormalities the patient had consistently were an anemia of chronic disease (hemoglobin, 90 to 120 g/L); an increased erythrocyte sedimentation rate (35 to 95 mm/h); an increased platelet count (350 to 726 109/L); increased levels of 1, 2, and globulins; and an increased complement C3 level (297 to 340 mg/dL [2.97 to 3.40 g/L]). The hallmark of the disease has been detection of a chemokinetic factor in the patients serum that caused wild random migration of purified normal human neutrophils and mononuclear leukocytes by up to 200%, which did not influence chemotaxis [1]. This factor has continued to be present during a period of 6 years despite various therapeutic attempts (see Table 1). The methods used to measure the leukocyte random migration and isolation of the serum factor have been previously described [1]. On 9 May 1991, after Food and Drug Administration and Local Investigational Review Board approvals (the patient gave informed consent), he was started on tacrolimus (FK 506), a new powerful immunosuppressive macrolide antibiotic [2]. He received an oral dose of 0.15 mg/kg twice daily. Twelve-hour tacrolimus trough plasma levels were measured with an enzyme-linked immunoassay [3]. Dose adjustments were guided by the drug plasma levels, the patients clinical response (skin and joint lesions), and the biochemical evidence of renal insufficiency (an increase of serum creatinine greater than 2 mg/dL) [4]. Because of the presence of a severe, disabling osteoporosis as well as recurrent vertebral compression and stress fractures, preexisting steroid therapy (20 to 60 mg/d) was rapidly tapered after starting tacrolimus treatment to a steroid dose of 5 mg orally every other day. All other therapeutic agents were discontinued before the initiation of tacrolimus therapy. Full dermatologic and medical examinations were done at each clinic visit. The assessments made included those quantifying pain, subcutaneous induration, erythema, cutaneous ulceration, arthritis, and drainage from the pyoderma gangrenosum lesions. Renal function, serum cholesterol, blood glucose, and electrolyte levels were also monitored. After 4 weeks, a marked reduction in his pain, lesion erythema, ulcer size, and drainage from lesions was evident. Complete clinical remission and healing of all disfiguring, large open sores was achieved after 12 weeks of tacrolimus (FK 506) therapy (Figure 1). Tacrolimus trough plasma levels were maintained at high levels for the initial 4 weeks (1.5 to 3.8 ng/mL) and subsequently at an average of 1.1 ng/mL. A recent effort to decrease his tacrolimus dosage resulted in low drug plasma levels (< 0.7 ng/mL) and a reappearance of focal subcutaneous areas of induration. These new lesions disappeared completely after an increase in the tacrolimus dose and maintenance of the drug plasma trough level at 1 ng/mL. Figure 1. Ulcer in patient with streaking leukocyte factor disease. Left. Right. The patient had transient trembling, paresthesias, and insomnia with high tacrolimus (FK 506) trough plasma levels. These were accompanied by increases in the serum creatinine and blood urea nitrogen levels to a maximum of 2.4 mg/dL and 51 mg/dL, respectively. A concomitant increase in blood pressure was treated with a calcium-channel blocker and a -adrenergic blocking agent. Intermittent hyperkalemia required treatment with fludrocortisone acetate (Florinef, Bristol-Myers Squibb, Somerville, New Jersey). Levels of serum creatinine, blood urea nitrogen, and serum potassium have stabilized at 1.9 mg/dL (168 mol/L), 28 mg/dL (10.0 mmol/L), and 4.9 mEq/L (4.9 mmol/L), respectively. No clinically significant change has occurred in the levels of blood glucose, cholesterol, or serum triglycerides. For the first time since birth, the patient does not have the skin lesions and the active joint disease. Moreover, he is now able to resume a full activity schedule. Six months after the start of tacrolimus (FK 506) treatment, a repeated assay of his serum for the streaking leukocyte factor showed an activity level equal to the control serum as well as the buffer solution, which contains 1% albumin and 10% fetal calf serum. The assay method used was identical to that used before tacrolimus therapy [1]. Because of the instability of the factor and other logistic problems, it has not been possible to do sequential studies in this patient to determine whether variations in the serum activity of the enhancing factor occur at different tacrolimus doses or drug plasma levels. Discussion The streaking leukocyte factor is a proteinaceous factor with a molecular weight of 160 kd that is found in the serum of some patients with recurrent sterile abscesses [1, 5]. It is thought to be responsible for progressive destruction and scarring of the skin and joints because it enhances random migration of polymorphonuclear and mononuclear leukocytes to areas of minor trauma. A fatal instance of this syndrome has been described recently [5]. Although the physical characteristics of this protein are not well known, it is too small to be an immunoglobulin and too large to be a classical cytokine. It is probably mutant secretory protein that has an abnormal effect on circulating leukocytes. The similarities and differences between this abnormal leukocyte-enhancing streaking factor and other chemokinetic factors present in normal serum, such as albumin and leukokinesis-enhancing factor, are described elsewhere [6]. The action of this abnormal leukocyte migration factor can be inhibited partially by corticosteroids and large doses of tetracycline [7-9]. The former agents may either block its action at a receptor site or, more likely, directly modify leukocyte responses so that they no longer react to the factor. Tetracycline probably acts as a nonspecific inhibitor of protein synthesis, limiting the production and secretion of the factor. Neither of these agents was successful in controlling the patients disease. Tacrolimus (FK 506) is a novel macrolide antibiotic isolated from the soil fungus Streptomyces tsukubaensis [2]. The drug has a wide range of immunosuppressive activities, because it inhibits the activation and proliferation of CD4+ T-helper lymphocytes [10]. The clinical experience during the previous 3.5 years has shown that a high therapeutic index exists for tacrolimus in patients who have transplanted organs [11] and for patients with either recalcitrant psoriasis or pyoderma gangrenosum [12, 13]. Previously there were encouraging results [13] after treating pyoderma gangrenosum; therefore, we decided to use tacrolimus for our patient. Interestingly, after using standard doses of tacrolimus in our patient (who had normal hepatic function), tacrolimus completely eliminated the streaking factor from our patients serum. This was associated with complete healing, although there is still scarring of the chronic skin ulcers, which had never resolved during the preceding three decades. The specific mode of action by which tacrolimus (FK 506) might alleviate this syndrome is unclear. However, the failure to detect the serum factor after treatment suggests that its synthesis or release or both was inhibited. This probably re-established the normal physiologic balance between the antagonistic activity of both the leukokinesis-enhancing factor and the cell-directed inhibitor [6]. Although it is not known whether tacrolimus has a direct inhibitory effect on leukocyte random mobility and chemotaxis, the drug appears to have no effect on lymphocyte chemoattractant-stimulated movement [14]. The adverse effects caused by tac

Combined kidney/bone marrow transplantation : evidence of augmentation of chimerism

Recent studies on patients who have maintained stable hepatic and renal allograft function for 10–28 years have revealed systemic microchimerism in all (1, 2). With the belief that this is the essential mechanism of allograft acceptance, we sought to augment the spontaneous chimerism in newly transplanted recipients, and began a program of combined simultaneous kidney/bone marrow transplantation without pretransplant radiation or other cytoreduction therapy (3). Postoperative immunosuppression was with FK506 and prednisone, using these drugs in the same way as in standard practice (4). Seventeen cadaver kidney recipients were entered into the bone marrow augmentation series up to June 1994. All are well. We report here the first 10, whose simultaneous kidney-bone marrow transplantations were between December 14, 1992 and December 14, 1993. Two of them also received pancreatic islets. The mean recipient age was 41.3 ± 13.7 years (range 24.6–63.4). All patients were undergoing their first transplantation and had a panel-reactive antibody level of less than 15%. The mean number of HLA matches and mismatches was 2.4 ± 1.3 (range 1–5) and 3.2 ± 1.8 (range 1–6), respectively. The mean donor age was 25.0 ± 7.5 years, and the mean cold ischemia time was 26.5 ± 9.4 hr. Eight control patients undergoing their first cadaveric kidney transplantation but not receiving bone marrow were also studied. The most common reason for not performing the combined procedure in the control cases was refusal of the donor family to consent to vertebral body recovery. Their mean age was 44.0 ± 11.0 years (range 29.7–59.8). The mean number of HLA matches and mismatches was 3.5 ± 1.9 (range 1–6) and 2.4 ± 2.0 (range 0–5). The mean donor age was 50.3 ± 12.4 years, and the mean cold ischemia time was 31.3 ± 3.6 hr. Neither irradiation nor any other kind of recipient preconditioning was used in the bone marrow recipients. All blood transfusions, if needed, were with irradiated packed red blood cells. After completion of the renal transplant procedure, 3 × 108 unmodified bone marrow cells/kg, isolated from the donor vertebral bodies, were infused via central line (3). Bone marrow infusion was performed at the time of closure of the transplant incision, or shortly thereafter in the recovery room. In the 2 patients who received islets, isolation was according to previously described techniques (5). The islets were infused into the portal vein, after completion of the kidney transplant. All of the protocols were approved by the Institutional Review Board of the University of Pittsburgh. Just prior to transplantation, 40 ml of blood was drawn and placed in heparinized tubes. Postoperatively, blood was drawn weekly for the first month, and monthly thereafter, for chimerism studies, which included fluorescent activated cell sorter analysis (FACS), polymerase chain reaction (PCR), and Y-chromosome analysis by both competitive PCR (cPCR) and fluorescent in-situ hybridization (FISH) of cytospin samples (in the case of female recipients of kidneys from the male donors). The mean follow-up for the 10 kidney/bone marrow recipients is 9.9 ± 4.7 months: all are alive and well, and all have functioning allografts. The mean serum creatinine and BUN are 1.6 ± 0.4 mg/dl, and 23 ± 8 mg/dl. Five (50%) patients have been weaned off steroids. The two islet recipients remain on insulin; they both have evidence of C-peptide production (0.44 and 0.11 pmol/ml), but at levels insufficient to allow for insulin independence. Two (20%) patients experienced early kidney nonfunction and required dialysis during the first postoperative week. The incidence of acute rejection was 50% (5 patients). All rejection episodes were responsive to steroids and/or an increase in the FK 506 dosage. No patient required antilymphocyte therapy. Cytomegalovirus (CMV) was seen in 2 (20%) patients and was treated successfully with intravenous gancyclovir. Graft vs. host disease (GVHD) was not seen any patient. The control recipients of a kidney only have been followed for 4.9 ± 0.5 months; all are alive and have functioning allografts (Table 1). The mean serum creatinine and BUN are 2.4 ± 1.0 mg/dl and 50 ± 32 mg/dl. None of these patients has been weaned off prednisone as of yet—however, this is most likely related to the relatively short follow-up. Two (25%) patients had acute tubular necrosis (ATN), although only 1 (13%) required dialysis during the first week after transplantation. The incidence of acute rejection was 63% (5 patients). All rejections were responsive to steroids and an increase in FK506. CMV was seen in 2 (25%) patients; both patients were successfully treated with gancyclovir. GVHD was not seen. TABLE 1 Summary of clinical course In the kidney/bone marrow patients, evidence of chimerism was present by at least one of the three modalities in 9 of 9 (100%) evaluable patients (Table 2). The tenth patient, who received a 1-B, 2-DR matched same-sex allograft, was not evaluable for microchimerism by any of these technologies. FACS was positive in 5 of 7 patients in whom evaluation was feasible, with a range of 0.9–3.0%. PCR for HLA disparities was positive in 8 of 8 patients in whom the study could be performed (Fig. 1). In the three female recipients of kidneys from male donors, Y-chromosome detection by PCR was positive in all cases and in one, competitive PCR (6) demonstrated a concentration of 0.5% donor DNA in PBMCs at 1 year. Using FISH for Y-chromosome detection, a level of 0.2–1.4% was seen (Fig. 2). FIGURE 1 Polymerase chain reaction (PCR) in a kidney/bone marrow recipient with a disparity for HLA-A29, demonstrating persistent chimerism. FIGURE 2 Fluorescent in-situ hybridization (FISH) of a cytospin preparation of PBMCs of a female recipient of a kidney/bone marrow from a male donor: orange represents X-chromosome; green represents Y-chromosome. TABLE 2 Testing for microchimerism In the kidney alone patients, 3 of the 8 patients could not be evaluated for microchimerism by any modality, because of complete DR matching and lack of donor sex disparity. In the 5 evaluable patients, 3 (60%) were positive for microchimerism by PCR, 1 by HLA disparity, and 2 by Y-chromosome analysis. However, FACS failed to detect microchimerism in any of 3 evaluable patients by 3 months after transplantation. Donor-specific reactivity could not be accurately assessed in 3 of the ten kidney/bone marrow recipients because there was complete DR-matching and a decreased in vitro immunologic response both pre- and post-transplantation. In the other 7 patients studied over time, there was one example of donor-specific nonresponsiveness and 2 of decreased donor-specific responsiveness that waxed and waned (Fig. 3). The other 4 patients retained donor-specific responsiveness. Only 5 of the 8 kidney-alone recipients were evaluable. One of the 5 has some evidence of decreasing responsiveness. FIGURE 3 Serial MLR responses of a kidney/bone marrow recipient. These results and those with 7 subsequently treated kidney-bone marrow recipients have shown that kidney/bone marrow transplantation is straightforward to perform and is safe. There were no examples of GVHD. Stable chimerism estimated to be 1000-fold more dense than that occurring spontaneously (3) was regularly achieved. It should be emphasized that (as was expected) bone marrow infusion did not influence the early events after transplantation. The incidence of acute rejection, the rates of early nonfunction, and the number cytomegalovirus infections were similar to those seen in patients receiving kidneys alone. However, treatment of these early problems was straightforward. After passing through this phase of convalescence, which was equally volatile with or without bone marrow augmentation, the more densely chimeric cohort of kidney-marrow recipients appeared to be an advantaged group. There was 100% patient and graft survival, and early completion of steroid weaning in half the cases. The full benefit of the chimeric state is expected to take one or 2 years, or longer. This trial was begun with patients undergoing primary transplantation who had low PRAs. We are now broadening our indications for kidney/bone marrow transplantation to include candidates undergoing retransplantation and/or those with high PRAs in order to assess the utility of adjuvant bone marrow in a more complex transplant setting. Historically important clinical attempts to facilitate kidney graft acceptance have been reported using adjunctive preoperative donor blood transfusions (7) or delayed (by 3 weeks) cryopreserved cadaveric donor bone marrow (8, 9). Unlike the premises of these earlier trials, our hypothesis was that long-term engraftment of the infused donor cells could occur without “making space” by host preconditioning, without an undue risk of GVHD, and using the same immunosuppressive strategy that fostered the previously unrecognized spontaneous development of microchimerism (1–3). The observations in the kidney recipients herein reported, as well as in recipients of livers and hearts (3) are confirmatory of these predictions.

LONG-TERM SURVIVAL OF DONOR-SPECIFIC PANCREATIC ISLET XENOGRAFTS IN FULLY XENOGENEIC CHIMERAS (WF RAT → B10 MOUSE)

We recently reported that reconstitution of lethally irradiated B10 mouse recipients with 40 x 10(6) untreated WF rat bone marrow cells resulted in stable fully xenogeneic chimerism (WF rat----B10 mouse). In these animals, the tolerance induced for skin xenografts was highly MHC specific in that donor-specific WF rat skin grafts were significantly prolonged while MHC-disparate third-party xenografts were rapidly rejected (median survival time [MST] = 9 days). We have now examined whether islet cell xenografts placed under the renal capsule of chimeras rendered diabetic with streptozotocin would be accepted and remain functional to maintain euglycemia. Animals were prepared, typed for chimerism at 6 weeks, and diabetes induced with streptozotocin. Donor-specific WF (Rt1Au) islet cell xenografts were significantly prolonged (MST greater than 180 days) in WF----B10 chimeras, while MHC-disparate third-party F344 rat (Rt1A1) grafts were rejected with a time course similar to unmanipulated B10 mice (MST = 8 days). The transplanted donor-specific islet cells were functional to maintain euglycemia, since removal of the grafts at from 100 to 180 days in selected individual chimeras uniformly resulted in return of the diabetic state. These data suggest that donor-specific islet cell xenografts are accepted and remain functional in mice rendered tolerant to rat xenoantigens following bone marrow transplantation.

Long-term (>3-Year) insulin independence in a patient with pancreatic islet cell transplantation following upper abdominal exenteration and liver replacement for fibrolamellar hepatocellular carcinoma

In the University of Pittsburgh experience, the most successful setting for human islet allografts is in patients undergoing upper abdominal exenteration with total pancreatectomy and liver transplantation for the indication of malignancy (cluster). In this group of patients 6/11 were insulin-independent for long periods. We report herein the metabolic course or the longest survivor (> 3 years). This patient has been free of exogenous insulin since the third postoperative month and has sustained her body weight without total parenteral nutrition since the 4th postoperative month. The patient has some postprandial hyperglycemia but average capillary glucoses are near-normal to normal as are glycosylated hemoglobin values. The clearance of glucose during the administration of an intravenous glucose load has been well preserved and is currently normal. C-peptide stimulates significantly in response to intravenously injected glucose. The absolute levels of stimulation during the test have declined possibly related to improvements in renal function, decreased immunosuppression or the natural history of cells transplanted into the portal site. The kinetics of the C-peptide response to intravenously injected glucose shows a persistent abnormality of first-phase insulin release and a prolonged second phase release. Basal glucagon levels are low but stimulate to a mixed meal. This patients results demonstrate long-term function of islet cells from a single donor transplanted into the portal vein using FK506 as an immunosuppressant agent.

Acceleration of chronic failure of intrahepatic canine islet autografts by a short course of prednisone

A topic of current interest in islet transplantation is the selection of an optimal site for long-term graft survival since the intrahepatic site may be characterized by long-term failure. Additionally, the use of immunosuppressive agents such as prednisone may adversely affect long-term graft function. In this study, we examined the long-term outcome of intrahepatic canine islet autografts and compared this with results obtained in animals treated with a short-term course of steroids or steroids plus insulin. Islets were isolated using the automated method and were purified on discontinuous Euro-Collins Ficoll gradients (densities: 1.108, 1.096, 1.037). Prednisone-treated dogs were hyperglycemic during treatment but returned to normoglycemia after steroid withdrawal. Control and insulin-treated animals were normoglycemic following autotransplant, with no difference in plasma glucose levels between controls and the insulin-treated animals. All control dogs became diabetic at 11, 14, 17, and 19 months following islet autograft. Prednisone-treated dogs had more rapid onset of diabetes at 7, 11, and 12 months following ITx. Prednisone-treated dogs given insulin became hyperglycemic at 10, 14, 18, and 19 months post ITx. Graft failure was preceded by a decline in IVGTT Kg values and diminished insulin secretion. At the time of graft failure islets showed no lymphocytic infiltration and islets stained positive for glucagon but few insulin-containing cells were seen. Thus, even when an initially adequate B cell mass was transplanted, the intrahepatic site was characterized by long-term canine autograft failure. A short course of prednisone accelerated the time to graft failure and insulin treatment reversed this acceleration.

EFFECT OF THE IMMUNOSUPPRESSANT FK506 ON GLUCOSE-INDUCED INSULIN SECRETION FROM ADULT RAT ISLETS OF LANGERHANS

The clinical use of cyclosporine has been a key advance in organ transplantation in the past decade (1). This agent is currently used in patients who receive all types of allografts. Also, CsA has been useful in preventing the onset of insulin-dependent diabetes in animal models (2) and inducing remissions in new-onset type I diabetes in humans (3). Despite the impressive achievements with cyclosporine, nephrotoxicity and graft rejection are still significant problems. Additionally, CsA decreases insulin synthesis and secretion in several species (4–7) and decreases glucose induced insulin secretion from human islets in vitro (8). It has been reported to decrease intravenous glucose tolerance when compared with azathioprine used in the same patient (9). The long-term consequences of these findings continue to be of concern. FK506 is a new immunosuppressive agent that is more potent on a weight basis than CsA (10). These drugs share many similar effects although the binding proteins for these two agents appear to be different. The binding sites share a novel isomerase enzyme system: peptidyl proline isomerase, which may represent an important common site of action (11). FK506 has recently been used successfully in trials in humans undergoing organ transplantation (12). In baboons, others have raised the issue of its potential adverse effect on glucose tolerance (13). Preliminary studies in human recipients of liver allografts showed that 6/12 patients had oral glucose tolerance tests that deviated from normal at at least one time point; 2 patients had impaired glucose tolerance, but only one patient had overt diabetes (14). These findings do not appear to differ from historical controls undergoing liver transplantation who did not receive FK506—however, the question of the effect of this agent on glucose metabolism remains an important unanswered question. FK506 apparently does not alter morphology or interfere with insulin secretion from fetal islets of Langerhans (15). Glucose-induced insulin secretion in neonatal and fetal islets is not comparable to the adult response (16, 17). Since adult tissues are often used in transplant studies, we looked at the effect of FK506 on glucose-induced insulin secretion from adult rat islets of Langerhans. We used freshly isolated islets obtained by collagenase digestion of the pancreas, from adult male Wistar rats (18). Insulin was measured by standard radioimmunoassay using a charcoal and dextran separation method (19). After isolation, islets were washed 5 times in basal (2.8 or 5.6 mM) glucose containing buffer.

The effect of FK506 on glycemic response as assessed by the hyperglycemic clamp technique.

We studied seven nondiabetic subjects with the autoimmune diseases psoriasis, multiple sclerosis, and primary biliary sclerosis who were to receive FK506 as experimental immunotherapy. All subjects underwent two standard oral glucose tolerance tests and two 180-min hyperglycemic clamps immediately before and 10 weeks after starting FK506. There was no significant difference in weight or HbA1c pre- vs. post-FK506 treatment. FK506 levels were therapeutic and non-toxic (0.1-1 ng/ml) for all subjects studied. Repeated measures analysis of variance for interaction between time and treatment was performed on insulin (after outlier removed) and glucose values from the OGTT. There was neither time-by-treatment interaction, nor a treatment effect (P > 0.1). There were no significant differences in pre- vs. post-FK506 treatment values of plasma glucose during the hyperglycemic clamp mean acute insulin response to glucose (AIRG) 164 +/- 38 pmol/L vs. 148 +/- 46 pmol/L (P > 0.1); mean incremental area under the insulin curve (IAUC) during the first 10 min of the study, 473 +/- 109 pmol/L vs. 443 +/- 146 pmol/L (P > 0.1); total area under the insulin curve (TAUC) during the first 10 min of the study, 786 +/- 152 pmol/L vs. 781 +/- 18 pmol/L (P > 0.1); mean glucose infusion rate (GIR) 37.7 +/- 5.0 mumol/kg/min vs. 33.3 +/- 4.4 mumol/kg/min (P > 0.1); or mean insulin sensitivity index (ISI), 3.05 +/- 0.4 vs. 3.13 +/- 0.5 (P > 0.1). Mean steady-state insulin secretion (SSI) was significantly lower 244 +/- 43 pmol/L vs. 200 +/- 25.2 pmol/L (P = 0.03). Peak first-phase insulin secretion values of 321 +/- 62 pmol/L vs. 263 +/- 57 pmol/L approached significance (P = 0.07). No patient progressed to diabetes during the study. FK506 decreased steady-state insulin secretion during the last 60 min of the clamp, regardless of initial glucose tolerance. Insulin sensitivity and glucose infusion rate did not change in the group as a whole with FK506 treatment.