Abdul S. Rao

Outcome analysis of 71 clinical intestinal transplantations

ObjectiveThe aim of the study was to determine risk factors associated with graft failure and mortality after transplantation of the intestine alone or as part of an organ complex. Summary Background DataEven with modern immunosuppressive therapies, clinical intestinal transplantation remains a difficult and unreliable procedure. Causes for this and solutions are needed. MethodsBetween May 1990 and February 1995, 71 intestinal transplantations were performed in 66 patients using tacrolimus and low-dose steroids. The first 63 patients, all but one treated 1 to 5 years ago, received either isolated grafts (n = 22), liver and intestinal grafts (n = 30), or multivisceral grafts (n = 11). Three more recipients of allografts who recently underwent surgery and one undergoing retransplantation were given unaltered donor bone marrow cells perioperatively as a biologic adjuvant. ResultsOf the first 63 recipients, 32 are alive: 28 have functioning primary grafts and 4 have resumed total parenteral nutrition after graft enterectomy. Thirty-five primary grafts were lost to technical and management errors (n = 10), rejection (n = 6), and infection (n = 19). Regression analysis revealed that duration of surgery, positive donor cytomegalovirus (CMV) serology, inclusion of graft colon, OKT3 use, steroid recycle, and high tacrlimus blood levels contributed to graft loss. All four intestine and bone marrow recipients are alive for 2–3 months without evidence of graft-versus-host disease. ConclusionTo improve outcome after intestinal transplantation with previous management protocols, it will be necessary to avoid predictably difficult patients, CMV seropositive donors, and inclusion of the graft colon. Bone marrow transplantation may further improve outcome by ameliorating the biologic barriers of rejection and infection and allowing less restrictive selection criteria.

Bone marrow augmentation of donor-cell chimerism in kidney, liver, heart, and pancreas islet transplantation

We have previously postulated that donor cell chimerism in organ transplantation is needed to attain a tolerant state. Here we show that donor cell chimerism can be augmented in organ recipients if they are infused perioperatively with 3 x 10(8) per kg of unmodified donor bone marrow cells and are kept on a conventional immunosuppressive regimen of tacrolimus (FK506) and prednisolone. 36 patients took part, of whom the first 18 patients have good transplanted kidney (n = 10), liver (n = 7), and heart (n = 7) function when followed up between 4 and 16 months. All patients are well. We found persistent multilineage leucocyte chimerism in blood of 17 recipients by flow cytometry and PCR techniques to detect donor alleles or Y chromosomes in female recipients of male organs. The use of the 5-antigben HLA matched same sex donor precluded detection of chimerism in one patient.

The lost chord: microchimerism and allograft survival

Recent evidence suggests that passenger leukocytes migrate after organ transplantation and produce persistent chimerism, which is essential for sustained survival of the allografts. Here, Thomas Starzl and colleagues argure that this hematolymphopoietic chimerism provides an important framework for the interpretation of basic and therapeutically oriented transplantataion research.

Clinical intestinal transplantation: new perspectives and immunologic considerations.

Background: Although tacrolimus-based immunosuppression has made intestinal transplantation feasible, the risk of the requisite chronic high-dose treatment has inhibited the widespread use of these procedures. We have examined our 1990–1997 experience to determine whether immunomodulatory strategies to improve outlook could be added to drug treatment. Study Design: Ninety-eight consecutive patients (59 children, 39 adults) with a panoply of indications received 104 allografts under tacrolimus-based immunosuppression: intestine only (n = 37); liver and intestine (n = 50); or multivisceral (n = 17). Of the last 42 patients, 20 received unmodified adjunct donor bone marrow cells; the other 22 were contemporaneous control patients. Results: With a mean followup of 32 ± 26 months (range, 1–86 months), 12 recipients (3 intestine only, 9 composite grafts) are alive with good nutrition beyond the 5-year milestone. Forty-seven (48%) of the total group survive bearing grafts that provide full (91%) or partial (9%) nutrition. Actuarial patient survival at 1 and 5 years (72% and 48%, respectively) was similar with isolated intestinal and composite graft recipients, but the loss rate of grafts from rejection was highest with intestine alone. The best results were in patients between 2 and 18 years of age (68% at 5 years). Adjunct bone marrow did not significantly affect the incidence of graft rejection, B-cell lymphoma, or the rate or severity of graft-versus-host disease. Conclusions: These results demonstrate that longterm rehabilitation similar to that with the other kinds of organ allografts is achievable with all three kinds of intestinal transplant procedures, that the morbidity and mortality is still too high for their widespread application, and that the liver is significantly but marginally protective of concomitantly engrafted intestine. Although none of the endpoints were markedly altered by donor leukocyte augmentation (and chimerism) with bone marrow, establishment of the safety of this adjunct procedure opens the way to further immune modulation strategies that can be added to the augmentation protocol.The advent of tacrolimus allowed clinical intestinal transplantation to become a feasible procedure for patients with irreversible intestinal failure. Over last 5 years, 71 patients underwent intestinal transplantation. Forty-one recipients were children, and 30 recipients were adults. Twenty-five patients received an isolated intestinal graft, 34 patients received a combined liver-intestinal graft, and 12 received a multivisceral graft. The colon was included the intestinal graft in 29 patients. One-year, 2-year, and 4-year actuarial patient survival is 72%, 57%, and 45%, respectively. Our experience has shown that infectious, and immunological problems have caused significant morbidity and mortality. In this paper, we present our clinical experience and overview with intestinal transplantation.

Interleukin-17 antagonism inhibits acute but not chronic vascular rejection.

BACKGROUND Blocking the action of interleukin (IL) 17 with an IL-17 receptor (R):Fc fusion protein inhibits T-cell proliferative responses to alloantigens and prolongs vascularized heart graft survival. In this study, we examined whether IL-17 antagonism could suppress the development of chronic rejection. METHODS A 0.6-cm section of C57BL10 (H2b) thoracic aorta was transplanted to recipient C3H (H2k) abdominal aorta. IL-17R:Fc or control human immunoglobulin G was administered i.p. (500 microg/day) from days 0 to 6 or from days 0 to 29. Mice were killed on days 7 or 30. Grafts were examined histologically and stained for alpha-smooth muscle actin (alpha-smA). Antidonor mixed leukocyte reaction, cytotoxic T cell, and alloantibody responses were quantified. RESULTS On day 7, control grafts showed mononuclear cell (MNC) infiltration, pronounced endothelial damage, and apoptosis of intimal and medial cell compartments. By day 30, there was concentric intimal thickening, accumulation of alpha-smA+ cells, and collagen deposition. Patchy destruction of the elastic membranes and loss of alpha-smA expression in media were evident. IL-17R:Fc for 6 days decreased MNC infiltration in the intimal and medial compartments at day 7. The endothelium was preserved (completely or partially) in all grafts. The medial compartment showed normal alpha-smA expression. Irrespective of IL-17R:Fc treatment for either 6 days or continuously, allografts harvested at day 30 showed circumferential intimal thickening, with accumulation of alpha-smA+ cells and collagen deposition. There was no effect on circulating alloantibody levels. CONCLUSIONS These findings support a role for IL-17 in the immunopathogenesis of acute vascular rejection and demonstrate the potential of IL-17 antagonism for therapy. By contrast, IL-17 antagonism does not appear to prevent ensuing chronic graft vascular disease, in particular neointimal formation.

Prevention of chronic rejection in mouse aortic allografts by combined treatment with CTLA4-Ig and anti-CD40 ligand monoclonal antibody

BACKGROUND In this study, using a murine model of aortic allotransplantation, the role of blockade of signaling through CD28/B7 and CD40/CD40 ligand costimulatory pathways in the evolvement of posttransplant vasculopathy was examined. METHODS Aortic allografts were transplanted across C57BL/1OJ (H2b)-->C3H (H2k) strain combinations. Transient or more stable blockade of second signaling was achieved by either a single injection or multiple injections of CTLA4-Ig fusion protein (200 microg/dose i.p.) and/or anti-CD40 ligand (CD40L) monoclonal antibody (250 microg i.m.). At day 30 after transplantation, the grafts were harvested for histopathological and immunohistochemical examination. RESULTS Similar to allografts of untreated animals, aortic allografts obtained from recipients treated with either CTLA4-Ig or anti-CD40L monoclonal antibody alone exhibited marked narrowing of the lumen primarily due to concentric intimal thickening caused by proliferation of alpha-smooth muscle actin-positive cells. Contemporaneous treatment, however, with either a single injection or multiple injections of CTLA4-Ig and anti-CD40L monoclonal antibody resulted in marked diminution of intimal thickening. Interestingly, concurrent prolonged inhibition of CD28/B7 and CD40/CD40L pathways resulted in complete abrogation of the development of posttransplant arteriopathy. CONCLUSION These data suggest that a more stable disruption of signaling through costimulatory pathways may be required to obviate the development of posttransplant vasculopathy.

ISOLATION, PHENOTYPE, AND ALLOSTIMULATORY ACTIVITY OF MOUSE LIVER DENDRITIC CELLS

Donor liver-derived dendritic cells (DC) have recently been identified within various lymphoid and nonlymphoid tissues of organ allograft recipients, including nonimmunosuppressed mice transplanted with and permanently accepting major histocompatibility complex (MHC)-disparate hepatic allografts. These findings have raised questions about the basis of the tolerogenicity of the liver--and, in particular, about the properties of liver-derived DC. To study further the structure, immunophenotype and allostimulatory activity of leukocytes resident in normal mouse (B10.BR;H-2k, I-Ek) liver, a procedure was developed to maximize the yield of viable, nonparenchymal cells (NPC) obtained following collagenase digestion of perfused liver fragments and density centrifugation (Percoll). These cells comprised populations expressing lymphoid and myeloid cell surface antigens. As compared with spleen cells, they proved good allostimulators of naive (B10; H-2b, I-E-) splenic T cells when tested in primary mixed leukocyte reactions (MLR). After overnight (18-hr) incubation of the NPC, enrichment for transiently adherent, low-density (LD) cells on metrizamide gradients permitted the recovery of low numbers of cells (approx. 2-5 x 10(5) per liver), many of which displayed distinct DC morphology. Flow cytometric analysis revealed that these cells were CD3-, CD4-, CD8-, and B220-, but strongly expressed CD45 (leukocyte-common antigen), and mild-to-moderate levels of CD11b, heat-stable antigen, and CD44. The cells also expressed moderate intensity of NLDC 145 but not 33D1, DC restricted markers which have been shown to be differentially expressed on mouse DC isolated from various organs. This DC-enriched population was more strongly MHC class II(I-Ek)+ than NPC, as determined by immunocytochemistry and flow cytometry and exhibited much more potent allostimulatory activity for naive T cells. These findings demonstrate that freshly isolated murine liver NPC, and perhaps their counterparts in situ, exhibit allostimulatory activity that is enhanced in the non-adherent, low-density (DC-enriched) fraction after overnight culture. They further suggest that the maturation of liver DC may play a key role in determining the immunogenicity and or tolerogenicity of hepatic allografts.

Autologous lymphokine-activated killer cell therapy of Epstein-Barr virus-positive and -negative lymphoproliferative disorders arising in organ transplant recipients

Lymphoreticular malignancies, collectively called posttransplant lymphoproliferative disorders (PTLD), eventually develop in 2-5% of organ transplant recipients. They frequently undergo regression when immunosuppression is reduced or stopped. This feature has been associated with a previous or de novo Epstein-Barr virus (EBV) infection. We herein describe immunotherapy with autologous lymphokine-activated killer (LAK) cells in seven patients with PTLD (four EBV-positive patients and three EBV-negative patients). Autologous peripheral blood mononuclear cells were obtained by leukapheresis, depleted of monocytes, and cultured in the presence of interleukin 2 for 10 to 11 days. A single dose of 5.2 x 10(9) to 5.6 x 10(10) LAK cells was given intravenously. Systemic interleukin 2 was not administered. The four patients with EBV+ PTLD had complete tumor regression; two of them developed controllable rejection. Three patients are well 13-16 months after treatment; the fourth patient died of pneumonia 41 days after infusion. Three patients with EBV- lymphomas had no response despite prior evidence that their tumors also were subject to immune surveillance. Two of these three patients died after being given other treatment, and the third patient has persistent tumor. In conclusion, autologous LAK cell infusion was effective for treatment of four EBV+ organ transplant recipients. LAK cell efficacy for three patients with EBV- PTLD was not evaluable under the management circumstances in which this treatment was utilized.

MULTILINEAGE HEMATOPOIETIC RECONSTITUTION OF SUPRALETHALLY IRRADIATED RATS BY SYNGENEIC WHOLE ORGAN TRANSPLANTATION: With Particular Reference to the Liver1

The migration of multilineage “passenger leukocytes” from transplanted organs and their ubiquitous survival in successfully treated human recipients for as long as 30 years (microchimerism) has been postulated to be an essential condition for organ allograft acceptance and the explanation for a number of previously enigmatic posttransplant events (1, 2). Following organ revascularization in either humans or rodents, donor leukocytes appear in large numbers in the recipient blood but usually diminish over a few days to a level undetectable with flow cytometry (2–5). We have construed the persistence of donor cells thereafter as prima facie evidence of the presence of hematopoietic stem and precursor cells (1,2,6). When the blood compartment is serially sampled in long-surviving human organ recipients, the levels of donor cells fluctuate (7), presumably reflecting cyclic activity of these stem cells. Taniguchi et al. (8) showed that primitive CD45+ cells purified from adult mouse livers unfailingly reconstituted all hematolymphopoietic lineages in supralethally irradiated adult mouse recipients. We present here direct evidence that supralethally irradiated adult rats can be consistently rescued by syngeneic liver transplantation, and that heart transplantation has a less dramatic but significant therapeutic effect. Inbred Lewis (RT11, LEW) rats weighing 200–250 g (8–10 weeks old) were purchased from Harlan Sprague Dawley (Indianapolis, IN) and kept in a laminar-flow specific-pathogen-free environment. Orthotopic liver and heterotopic (abdominal) heart transplantation were performed as described previously (5). After death of the donor by exsanguination, the grafts were flushed intravascularly with chilled lactated Ringer’s solution until the venous effluent was clear. Bone marrow cells were harvested from the tibias and femurs, and processed in RPMI 1640 supplemented with 25 mM Hepes buffer, 2 mM L-glutamine, 50 U/ml penicillin, and 50 μg/ml streptomycin (all from Gibco, Grand Island, NY) (5). Trypan blue exclusion testing uniformly showed >95% cell viability. The cell suspensions were administered into the recipient penile vein. A reagent panel containing monoclonal antibodies against all of the principal leukocyte subsets was used to determine lineages of the control and reconstituted animals (panel available on request). Most experiments were performed with male donors and recipients. However, in 2 liver recipients, male → female transplantation was performed and the tissues and organs of the hematopoietically reconstituted recipient were studied with polymerase chain reaction and Southern hybridization, using rat Y-chromosome (sex determining region Y [SRY]) specific primers (9). This provided proof of hematopoietic sex change in the reconstituted animals as opposed to recovery of the cytoablated recipient stem cells. Recipients were irradiated with 9.5 Gy, delivered from a cesium source. All cell or organ transplantations were done 4–8 hr after completion of irradiation. Survival was determined of animals receiving 4 different doses of syngeneic bone marrow, and of those given liver and heart grafts (Table 1). In addition, 6 irradiated animals of similar size were given 3 ml of fresh whole blood, which was an estimated 25% of their total blood volume. This amount of transfusion exceeded by an estimated thousand-fold or more any residual blood in the thoroughly flushed organs used for transplantation. Table 1 Survival of lethally irradiated (9.5 Gy) LEW rats after different types of syngeneic organ and cell transplantation All untreated animals died within 12 days. Heart transplantation significantly prolonged survival (P<0.05). One of 6 cardiac recipients was permanently rescued and 4 of the 5 others lived 2–6 days beyond the longest surviving non-treated control. These results were at least as good as those obtained with a suboptimal dose of 0.5×106 bone marrow cells (P=0.109 vs. control) and with the infusion of 3 ml of whole blood (P=0.007 vs. control). Liver transplantation resulted in permanent survival of 5 of 6 irradiated recipients with full multilineage reconstitution, a rescue effect that was not significantly different from that following infusion of 1,5, or 10×106 bone marrow cells (Table 1). Because frequent blood sampling and other manipulations jeopardized survival, hematologic studies were done in a separate cohort of irradiated animals, with subgroups that were untreated, infused with 1×106 or 1×107 unmodified bone marrow cells, or submitted to liver transplantation. The results, including weight gain, were compared with those in naive unaltered animals in the same environment (Figs. 1–3). Figure 1 Total white cell counts (mm3) in normal and irradiated LEW rats (9.5 Gy) and rats given syngeneic transplants, after treatment with red blood cell lysing buffer. Cells were counted in a hemacytometer. ●, Normal Lewis (n=3); ○, radiation ... Figure 3 Body weight changes in animals of Figures 1 and ​and2.2. ●, Normal LEW (n=3); ○, radiation alone (n=3); ■ 1×106 bone marrow (n=6); □, 10×106 bone marrow (n=4); ▲ OLTx (n=3). The rate and the extent of circulating leukocyte (Fig. 1) and hematocrit restoration (Fig. 2) were similar after liver transplantation and infusions of 1×106 bone marrow cells. Rats given a 10-fold higher dose of bone marrow cells (1×107) recovered more quickly (Figs. 1 and ​and2).2). The sequence of lineage recovery was similar in the liver and bone marrow recipients, and appeared to be dependent on the number of progenitor cells. The FACS profile of animals after various kinds of hematopoietic reconstitution is shown in Figure 4. The reconstituted cells in cross-sex liver transplantations (male → female) were predominantly donor (Fig. 5) and were, of course, least densely represented in the heart. Figure 2 Hematocrit (tail vein blood, microcentrifuge method) in same samples as Figure 1. Figure 4 Lineage of peripheral leukocytes shown in Figure 1. Granulocytes were determined on blood smear following α-naphthyl acetate esterase and hematoxylin staining. Other FACS analysis of percentage of subpopulations (mean ± SD) was by direct ... Figure 5 Polymerase chain reaction and Southern hybridization of organs and tissues 50 days after lethal irradiation and syngeneic liver transplantation. PBL, peripheral blood lymphocytes; Ty, thymus; SP, spleen; BM, bone marrow; CLN, cervical lymph nodes. Male ... The initial delay in postoperative weight gain of liver compared with bone marrow recipients (Fig, 3) was explained by the greater stress and metabolic depletion associated with the hepatic replacement compared with the less traumatic penile vein infusion of bone marrow cells. After 3 weeks, the liver recipients had catch-up weight gain, and they achieved parity with all other reconstituted groups by day 50 (Fig. 3). These experiments suggest that the difference between the chimerism (and tolerance) following classical bone marrow transplantation and that produced by the donor (“passenger”) leukocytes of whole organs is purely semantic. While describing the multilineage character of the microchimerism in human and animal organ recipients (1, 2, 6, 10, 11), we have emphasized the invariable histopathologic prominence of dendritic cells because their presence was so much in conflict with the literature preceding 1992. The previous literature had associated these cells almost exclusively with rejection rather than graft acceptance. We have recently summarized elsewhere the impressive evidence acquired since then of a key tolerogenic role played by the dendritic cells, particularly when in their precursor stages (12). However, preoccupation with a single leukocyte lineage could obscure an understanding of the complex cell interactions that we are seeking to understand and manipulate. We believe it unlikely that transplantation tolerance can be fully explained, reliably induced, or efficiently sustained by any single lineage (5, 6, 13). In the experiments reported herein, heart transplantation also had a significant effect on postirradiation survival, less dramatic than the liver but similar to that of a suboptimal dose of donor bone marrow or a large blood transfusion. The permanent hematopoietic reconstitution of one of the heart recipients and prolongation of survival of 4 of 5 others strengthen our earlier contention that tolerogenicity is an inherent capability common to all organized tissues and organs, with variations in outcome dictated by the quantity and lineage profile of bone marrow-derived leukocytes contained in the graft (1, 2, 5, 6, 13). No record could be found in the literature suggesting that these simple and direct reconstitution experiments had been performed previously, presumably because of the assumption that organ transplantation involves a unidirectional host-versus-graft immune reaction (the one-way paradigm). Leszczynski et al. (14) showed that 1×106 recipient inflammatory cells extracted from rejecting rat kidney allografts contained enough host stem cells to fully reconstitute supralethally irradiated animals of the recipient strain. The experiments reported herein showed that the transplanted organ (especially the liver) brings enough donor stem cells to provide the basis of the sustained bidirectional immune reaction that we have postulated to be the basis of allograft acceptance and the usual form of transplantation tolerance (the two-way paradigm [5, 15]).

Enhanced synthesis and reduced metabolism of endothelin-1 (ET-1) by hepatocytes - an important mechanism of increased endogenous levels of ET-1 in liver cirrhosis

BACKGROUND/AIMS Hepatic concentration of endothelin-1 (ET-1) is increased in human and experimental liver cirrhosis. Because of its potent actions in the liver, ET-1 has been suggested to play an important role in the pathophysiology of cirrhosis. Since hepatocytes are the major cell type to metabolize ET-1, we investigated whether their reduced capacity to degrade ET-1 is a mechanism of its elevated levels in cirrhosis. METHODS The expression of ET-1 receptors, ET-1 and endothelin converting enzyme (ECE), and metabolism of ET-1 and ECE activity were compared in hepatocytes isolated from control and carbon tetrachloride-induced cirrhotic rats. RESULTS ET-1 receptor density and receptor-mediated internalization of ET-1 were significantly increased in cirrhotic hepatocytes relative to the control cells. However, compared to control hepatocytes, metabolism of ET-1 by the cirrhotic cells was reduced significantly. Interestingly, hepatocytes were found to contain preproET-1 mRNA, ECE-1 mRNA and ET-1. PreproET-1 mRNA and ET-1 levels were increased in cirrhotic hepatocytes but their ECE mRNA and ECE activity were not altered. CONCLUSIONS These results provide the first evidence that hepatocytes have the ability to synthesize ET-1 and demonstrate that decreased metabolism and enhanced synthesis, of ET-1 in hepatocytes are an important mechanism of its elevated levels in cirrhosis.