Ajai Khanna

Effects of Liver-Derived Dendritic Cell Progenitors on Th1- and Th2-Like Cytokine Responses In Vitro and In Vivo

There is evidence that donor-derived dendritic cells (DC), particularly those at a precursor/immature stage, may play a role in the immune privilege of liver allografts. Underlying mechanisms are poorly understood. We have examined the influence of in vitro generated mouse liver-derived DC progenitors (DCp) on proliferative, cytotoxic, and Th1/Th2 cytokine responses induced in allogeneic T cells. Liver DCp, propagated in GM-CSF from C57B10 mice (H2b), induced only minimal proliferation, and weak cytotoxic responses in allogeneic (C3H; H2k) T cells compared with mature bone marrow (BM)-derived DC. Flow-cytometric analysis of intracellular cytokine staining revealed that mature BM DC, but not liver DCp, elicited CD4+ T cell production of IFN-γ. Intracellular expression of IL-10 was very low in both BM DC- and liver DCp-stimulated CD4+ T cells. Only stimulation by liver DCp was associated with IL-10 secretion in primary MLR. Notably, these liver DCp cocultured with allogeneic T cells stained strongly for IL-10. Following local (s.c.) injection in allogeneic recipients, both BM DC and liver DCp homed to T cell areas of draining lymph nodes and spleen, where they were readily detected by immunohistochemistry up to 2 wk postinjection. Liver DCp induced clusters of IL-10- and IL-4-secreting mononuclear cells, whereas Th2 cytokine-secreting cells were not detected in mice injected with mature BM DC. By contrast, comparatively high numbers of IFN-γ+ cells were induced by BM DC. Modulation of Th2 cytokine production by donor-derived DCp may contribute to the comparative immune privilege of hepatic allografts.

LONG-TERM RESULTS OF PANCREAS TRANSPLANTATION UNDER TACROLIMUS IMMUNOSUPPRESSION

Background The long-term safety and efficacy of tacrolimus in pancreas transplantation has not yet been demonstrated. The observation of prolonged pancreatic graft function under tacrolimus would indicate that any potential islet toxicity is short-lived and clinically insignificant. We report herein the results of pancreas transplantation in patients receiving primary tacrolimus immunosuppression for a minimum of 2 years. Methods. From July 4, 1994 until April 18, 1996, 60 patients received either simultaneous pancreas-kidney transplant (n=55), pancreas transplant only (n=4), or pancreas after kidney transplantation (n=1). Baseline immunosuppression consisted of tacrolimus and steroids without antilymphocyte induction. Azathioprine was used as a third agent in 51 patients and mycophenolate mofetil in 9. Rejection episodes within the first 6 months occurred in 48 (80%) patients and were treated with high-dose corticosteroids. Antilymphocyte antibody was required in eight (13%) patients with steroid-resistant rejection. Results. With a mean follow-up of 35.1±5.9 months (range: 24.3-45.7 months), 6-month and 1-, 2-, and 33-year graft survival is 88%, 82%, 80%, and 80% (pancreas) and 98%, 96%, 93%, and 91% (kidney), respectively. Six-month and 1-, 2-, and 3-year patient survival is 100%, 98%, 98%, and 96.5%. Mean fasting glucose is 91.6±13.8 mg/dl, and mean glycosylated hemoglobin is 5.1±0.7% (normal range: 4.3-6.1%). Mean tacrolimus dose is 6.5±2.6 mg/day and mean prednisone dose 2.0±2.9 mg/day at follow-up. Complete steroid withdrawal was possible in 31 (65%) of the 48 patients with functioning pancreases. Conclusions. These data show for the first time that tacrolimus is a safe and effective long-term primary agent in pancreas transplantation and provides excellent long-term islet function without evidence of toxicity while permitting steroid withdrawal in the majority of patients.

Noncompliance after pediatric liver transplantation.

There were 34 episodes of documented noncompliance among 28 patients (15 males, 13 females) transplanted between March 1984 and May 1993. The mean age at the time of transplantation was 9 years (range, 2.6 to 16 years). The first episode of noncompliance was diagnosed at a mean age of 14.8 years (range, 10 to 20 years), and a mean of 6.5 years (range, 1.4 to 11.9 years) after transplantation. In those cases in which there was more than one episode of noncompliance, the mean time from the first to the second episode was 2.1 years (range, 0.6 to 3.9 years). At the time of noncompliance, 11 patients were on cyclosporine A (CyA) and 17 were receiving FK 506 (9 of which had been previously converted from CyA). There was a mean of 2.19 (range 0 to 6) episodes of rejection per patient before the diagnosis of noncompliance. Seventy-nine percent (n 5 22) of the noncompliant children had a history of psychiatric/ psychosocial associated factors. Liver biopsy specimens obtained at the time of noncompliance in 18 patients showed acute cellular rejection (n 5 8), hepatitis (n 5 5), hepatitis/rejection (n 5 3), and pericholangitis (n 5 2). Twenty-two patients had reinstitution of their baseline immunosuppressive drugs, FK 506 (n 5 17), and CyA (n 5 5). The remaining six were converted from CyA to FK 506. Four grafts were lost to chronic rejection, and one to rupture of a hepatic artery aneurysm after successful treatment of rejection. Three patients required retransplantation, one of them twice. There were two recurrences of noncompliance after retransplantation. Two patients died, the patient with the ruptured aneurysm and one of the retransplanted patients secondary to chronic rejection.

LIVER TRANSPLANTATION FOR METABOLIC LIVER DISEASES

Liver transplantation has revolutionized the outcome of metabolic liver diseases that are caused by defects in hepatocytes (e.g., Wilsons disease) or by excessive deposition of substrates secondary to their increased absorption (e.g., hemochromatosis). Early diagnosis and referral are the keys to successful outcome. The timing of liver transplantation for patients on medical therapy depends on a lack of biochemical and clinical evidence of improvement. Overall outcome following liver transplantation depends on the severity of multisystem involvement and preoperative decompensation.

Reasons for long-term use of steroid in primary adult liver transplantation under tacrolimus.

Background. Tacrolimus is a potent immunosuppressive agent that provides higher freedom from acute and chronic rejection than cyclosporine after liver transplantation (LTx). Initially, a steroid-free state was observed in about 70% of patients at 1 year; this did not change over the next 5 years. The present study identifies the various reasons why the remaining 30% of adult patients still require steroids even after 5 years after successful LTx. Method. Eight hundred thirty-four consecutive patients who underwent LTx between August 1989 and December 1992 were included in this study. Four hundred ninety-nine patients were alive in January 1999 and were available for this study. The dose of steroid and the reason for steroid use were retrospectively determined from the clinical records. Results. Three hundred sixty-five patients (73.1%) were off steroid, whereas 134 patients (26.9%) were receiving prednisone (mean dose was 6.4±3.7 mg/day) at the time of the study. Four hundred and eight-four patients (97%) were off prednisone at some time after LTx; however, in 119 (23.8%) patients, steroids were reintroduced. Fifteen patients (3%) continued to receive prednisone; eight receive prednisone due to reluctance of the local physician to withdraw the medication; in five patients, the prednisone was not withdrawn because these patients were on cyclosporine; in the remaining two patients, repeated attempts to withdraw steroid resulted in a rise in liver function test. In the 49 (36.6%) of 119 patients in whom the steroid was reintroduced, it was restarted secondary to pathologically proven or clinically suspected rejection (group I). In five patients steroid was reintroduced for abnormal liver function after being off immunosuppression for treatment of a posttransplantation lymphoproliferative disorder. Six patients were noncompliant with their immunosuppressive medication, and the steroid was reintroduced to control rejection. Steroids were reintroduced in 30 patients (22.4%) for recurrence of original disease: primary biliary cirrhosis (n=19), sclerosing cholangitis (n=6), and autoimmune hepatitis (n=5) (group II). In 24 patients (20.2%), steroids were reintroduced to lower the dose of tacrolimus secondary to nephrotoxicity. Six of these patients received kidney transplantation (group III). In 16 patients (13.4%) the steroid was reintroduced for concomitant medical problems, consisting of ulcerative/Crohn’s colitis (n=6), adrenal insufficiency (n=5), hematological disorders (n=3), dermatitis (n=1), and rheumatoid arthritis (n=1) (group IV). Conclusion. Ninety-seven percent of patients under tacrolimus were weaned off steroid; however, 23.8% required steroid reintroduction for late rejection, recurrence of autoimmune process(es), renal impairment, or the concomitant presence of other medical conditions. Although the use of other immunosuppressive agents may reduce the rate of reintroduction of steroid, long-term sustained freedom from steroid may not be possible in all patients under tacrolimus secondary to these conditions.

IMMUNOSUPPRESSIVE THERAPY: New Concepts

Although several new immunosuppressive medications have been developed in the past decade, many possible avenues are yet to be explored. Although the newer agents have not reflected any clear benefit in patient or graft survival over CsA or tacrolimus, they have been useful in reducing the incidence and severity of rejection, reducing the concomitant use of steroids, and decreasing the doses of CsA or tacrolimus to minimize their toxicity profile. The appearance of these new agents has given more options to clinicians, who can select the one with the least toxicity and most efficacy for individual patients. In the future, combinations of these agents, in conjunction with a strategy to induce tolerance of the donor organ without drug toxicity, will be the goal.

Cardiac changes at autopsy in adult liver transplant recipients under tacrolimus

Tacrolimus (FK506) is being used as immunosuppressant in solid organ transplantation. However, its nephrotoxic and neurotoxic effects are well recognized. Recently reversible hypertrophic cardiomyopathy under tacrolimus has been described in five children.1 In order to evaluate myocardial toxicity of the drug, we examined the cardiac findings at autopsy in adults and children following primary liver transplantation (OLTx) under tacrolimus and compared them with autopsy findings of patients who died of end-stage liver disease without OLTx and tacrolimus.

FLT-3 LIGAND INCREASES MICROCHIMERISM BUT CAN PREVENT THE THERAPEUTIC EFFECT OF DONOR BONE MARROW IN TRANSIENTLY IMMUNOSUPPRESSED CARDIAC ALLOGRAFT RECIPIENTS

C3H (H2k) mice received 50 x 10(6) B10 (H2b) bone marrow (BM) cells either alone or with flt-3 ligand (FL) (10 microg/day), tacrolimus (2 mg/kg/day), or both agents for 7 days. Donor MHC class II+ (IAb+) cells were quantitated in spleens by immunohistochemical analysis, and donor class II DNA detected in BM by PCR. Donor cells were rare in the BM alone and BM + FL groups, whereas there was a substantial increase in chimerism in the BM + tacrolimus group. Addition of FL to BM + tacrolimus led to a further eightfold increase in donor cells and enhanced donor DNA compared with the BM + tacrolimus group. This increase in donor cells was almost 500-fold compared with BM alone. C3H recipients of B10 heart allografts given perioperative B10 BM and tacrolimus (days 0-13) exhibited a markedly extended median graft survival time (MST, 42 days) compared with those given tacrolimus alone (MST, 22 days). Addition of FL (10 microg/day; 7 days) to BM + tacrolimus prevented the beneficial effect of donor BM (MST, 18 days). BM alone or BM + FL resulted in uniform early heart graft failure (MST < 8 days). Functional studies revealed maximal antidonor MLR and CTL activities in the BM- and BM + FL-treated groups, with minimal activity in the tacrolimus-treated groups. Thus, dramatic growth factor-induced increases in chimerism achieved under cover of immunosuppression may result in augmented antidonor T cell reactivity and reduced graft survival after immunosuppressive drug withdrawal. With FL, this may reflect striking augmentation of immunostimulatory dendritic cells.

Impact of Flt-3 ligand on donor-derived antigen presenting cells and alloimmune reactivity in heart graft recipients given adjuvant donor bone marrow

The influence of the haematopoietic growth factor Flt-3 ligand (FL) on the incidence and function of donor major histocompatibility complex (MHC) class II+ cells in the lymphoid tissues of noncytoablated recipients of heart allografts and donor bone marrow (BM) cells was investigated. C3H (H2k) mice received a nonvascularized B10 (H2b) heart allograft in the dorsal ear pinna, followed by an i.v. infusion of 50 x 10(6) donor BM cells. They were given FL (10 microg/day i.p., x7 days), tacrolimus (2mg/kg/day i.p., x13 days) or both agents immediately following heart transplantation (HTx) and were killed 10 or 21 days later. Their BM cells were propagated in vitro in granulocyte macrophage colony stimulating factor (GM-CSF) and IL-4 for 5 days to promote the growth of dendritic cells (DC). Donor DC were identified by immunocytochemical staining. Spleens were harvested, and donor (IAb+) cells enumerated by immunohistochemical analysis. Donor MHC class II DNA was detected in spleens and cultured BM-derived cells by reverse transcriptase-polymerase chain reaction (RT-PCR). A striking increase in donor MHC class II+ cells was noted in both the spleen and BM of the BM + tacrolimus-treated group compared to either the BM alone, or BM + FL-treated groups. Addition of FL treatment to BM + tacrolimus led to a further increase in donor cells in spleen (three-fold at 10 days, and two-fold at 21 days). The increase in donor cells at 10 days was almost 140-fold compared to that with donor BM alone. PCR analysis at this time revealed enhanced donor DNA in the BM + FL + tacrolimus group compared to that in the BM + tacrolimus group. FL treatment augmented mixed leucocyte reactions (MLR) and cytotoxic T lymphocyte (CTL) activity of host spleen cells against donor alloantigens. These effects were reversed by tacrolimus administration. Histopathology of heart grafts from tacrolimus-treated animals at 10 and 21 days showed absence or substantial reduction in cellular infiltration, and the preservation of viable myocardium. By contrast, in untreated mice, or animals given BM or BM + FL alone, there was marked cellular infiltration, and features of accelerated rejection. Donor-derived DC could be propagated in vitro from the BM of heart transplant recipients given donor BM, especially from mice that also received tacrolimus +/- FL. At day 21, donor-derived cells could only be propagated from the BM + FL + tacrolimus-treated group. These findings show that numbers of donor antigen presenting cells (APC) or their progenitors can be markedly increased in conventionally immunosuppressed organ allograft recipients given donor BM + a potent haematopoietic and DC-growth promoting cytokine. Although withdrawal of systemic immunosuppression appears to allow exhibition of the potential allostimulatory activity of these donor APC leading to rejection, the model provides a useful basis for further evaluation of the persistence and manipulation of donor haematopoietic cells and in particular, donor-derived APC, on the outcome of organ transplantation.

Improvements in intestine transplantation

Transplantation of the intestine in children has presented significant challenges even as it has become a standard to treat nutritional failure due to short gut syndrome. These challenges have been addressed in part by significant improvements in short and long-term care. Noteworthy enhancements include reduced need for intestine transplantation, drug-sparing immunosuppressive regimens, immune monitoring, and improved surveillance and management of PTLD and non-adherence.