A Double-Blind, Double-Dummy, Flexible-Design Randomized Multicenter Trial: Early Safety of Single- Versus Divided-Dose Rabbit Anti-Thymocyte Globulin Induction in Renal Transplantation.

A previous nonblinded, randomized, single-center renal transplantation trial of single-dose rabbit anti-thymocyte globulin induction (SD-rATG) showed improved efficacy compared with conventional divided-dose (DD-rATG) administration. The present multicenter, double-blind/double-dummy STAT trial (Single dose vs. Traditional Administration of Thymoglobulin) evaluated SD-rATG versus DD-rATG induction for noninferiority in early (7-day) safety and tolerability. Ninety-five patients (randomized 1:1) received 6 mg/kg SD-rATG or 1.5 mg/kg/dose DD-rATG, with tacrolimus-mycophenolate maintenance immunosuppression. The primary end point was a composite of fever, hypoxia, hypotension, cardiac complications, and delayed graft function. Secondary end points included 12-month patient survival, graft survival, and rejection. Target enrollment was 165 patients with an interim analysis scheduled after 80 patients. Interim analysis showed primary end point noninferiority of SD-rATG induction (p = 0.6), and a conditional probability of <1.73% of continued enrollment producing a significant difference (futility analysis), leading to early trial termination. Final analysis (95 patients) showed no differences in occurrence of primary end point events (p = 0.58) or patients with no, one, or more than one event (p = 0.81), or rejection, graft, or patient survival (p = 0.78, 0.47, and 0.35, respectively). In this rigorously blinded trial in adult renal transplantation, we have shown SD-rATG induction to be noninferior to DD-rATG induction in early tolerability and equivalent in 12-month safety. (Clinical Trials.gov #NCT00906204.).

Increased primary non-function in transplanted deceased-donor kidneys flushed with histidine-tryptophan-ketoglutarate solution.

Histidine-Tryptophan-Ketoglutarate (HTK) solution is increasingly used to flush and preserve organ donor kidneys, with efficacy claimed equivalent to University of Wisconsin (UW) solution. We observed and reported increased graft pancreatitis in pancreata flushed with HTK solution, which prompted this review of transplanting HTK-flushed kidneys. We analyzed outcomes of deceased-donor kidneys flushed with HTK and UW solutions with a minimum of 12 months follow-up, excluding pediatric and multi-organ recipients. We evaluated patient and graft survival and rejection rates, variables that might constitute hazards to graft survival and renal function. Two-year patient survival, rejection, renal function and graft survival were not different, but early graft loss (<6 months) was worse in HTK-flushed kidneys (p < 0.03). A Cox analysis of donor grade, cold ischemic time, panel reactive antibodies (PRA), donor race, first vs. repeat transplant, rejection and flush solution showed that only HTK use predicted early graft loss (p < 0.04; relative risk = 3.24), almost exclusively attributable to primary non-function (HTK, n = 5 (6.30%); UW, n = 1 (0.65%); p = 0.02). Delayed graft function and early graft loss with HTK occurred only in lesser grade kidneys, suggesting it should be used with caution in marginal donors.

Increased pancreatitis in allografts flushed with histidine-tryptophan-ketoglutarate solution: a cautionary tale.

We reviewed pancreas transplantation outcomes after Histidine-Tryptophan-Ketoglutarate (HTK) and University of Wisconsin (UW) preservation solution use between 2001 and 2007 at two transplant centers. While equivalence has been claimed for kidney and liver transplant outcomes after the use of HTK or UW preservation solution, consensus has not been reached on equivalence when flushing pancreata. Others have reported comparable patient and graft survival rates, but found an association between the use of HTK and an increase in the incidence of acute rejection and pancreatitis. In reviewing our experiences, we found in pancreata flushed with HTK a higher incidence of postoperative complications including graft pancreatitis, use of octreotide and a decreased rate of insulin-independence at hospital discharge. These findings prompted us to critically review our centers' experience to determine if there is a basis for suspecting a causal relationship.

Hashimoto's encephalopathy after intensive lymphocyte depletion with rabbit anti-thymocyte globulin in a renal transplant patient.

There has been no reported case of Hashimoto's encephalopathy (HE) ('steroid-responsive encephalopathy associated with autoimmune thyroiditis', 'SREAT'), in the renal transplant recipient population. We describe the case of a 55-year-old female with Type-1 diabetes who presented 2 years posttransplantation in a comatose state that had developed over the preceding 24 h. The patient had received a short, intensive course of rATG induction at the time of transplantation and early steroid withdrawal. After 6 months she had been withdrawn from calcineurin inhibitors and was maintained on mycophenolate mofetil and sirolimus. Systematic workup determined the cause of her coma to be HE. High-dose steroid therapy resulted in complete resolution of the patient's symptoms. The literature regarding the diagnosis, course and treatment of HE is reviewed and the possibility that increased use of steroid-free immunosuppression and intensive lymphocyte depletion regimens may increase the prevalence of de novo autoimmune disease is discussed.

Modulation of immune responses after portal venous injection of antigen.

BACKGROUND: How the localization of antigen to the liver, through means such as oral ingestion, induces tolerance is poorly understood. METHODS: To elucidate potential mechanisms we used an adoptive transfer system wherein ova-specific T cells were infused into a syngeneic host, and antigen-specific T-cell responses after delivery of soluble antigen into the liver were monitored. RESULTS: After infusion of antigen into the portal vein, the frequency of antigen-specific T cells in lymph nodes draining the liver was lower than the frequency in peripheral lymph nodes. These findings were the reverse of what is typically observed after subcutaneous injection of antigen with adjuvant. Infusion of antigen with adjuvant into the portal vein did not alter this pattern of antigen-specific T-cell localization; however, an increased frequency of T cells, compared with antigen alone, was observed in peripheral lymph nodes and spleen. After exposure to antigen via the portal vein, T cells isolated from lymph nodes draining the liver and challenged with antigen in vitro exhibited a diminished proliferative response compared with T cells isolated from nondraining lymph nodes. This hyporesponsiveness was not observed when the antigen was administered with adjuvant. CONCLUSIONS: Our findings suggest that the influence of the liver on immune responses might reflect two processes: (1) loss of antigen-specific T cells after primary antigen injection, and (2) hyporesponsiveness on reexposure to antigen. These mechanisms may contribute to the prevention of undesirable immune responses to foods and enteric bacteria in the gastrointestinal tract. Furthermore, these results underscore the importance of minimizing inflammation in circumstances such as islet transplantation, if endogenous mechanisms of tolerance induction are to be maximized.

Phenotypic and functional maturation of dendritic cells mediated by heparan sulfate.

Primary immune responses are thought to be induced by dendritic cells. To promote such responses, dendritic cells must be activated by exogenous agonists, such as LPS, or by products of activated leukocytes, such as TNF-alpha and IL-1. How dendritic cells might be activated in the absence of exogenous stimuli, or without the immediate presence of activated leukocytes, as might occur in immunity to tumor cells or transplants, is unknown. We postulated that heparan sulfate, an acidic, biologically active polysaccharide associated with cell membranes and extracellular matrices, which is rapidly released under conditions of inflammation and tissue damage, might provide such a stimulus. Incubation of immature murine dendritic cells with heparan sulfate induced phenotypic maturation evidenced by up-regulation of I-A, CD40, CD54 (ICAM-1), CD80 (B7-1), and CD86 (B7-2). Dendritic cells exposed to heparan sulfate exhibited a markedly lowered rate of Ag uptake and increased allostimulatory capacity. Stimulation of dendritic cells with heparan sulfate induced release of TNF-alpha, IL-1beta, and IL-6, although the maturation of dendritic cells was independent of these cytokines. These results suggest that soluble heparan sulfate chains, as products of the degradation of heparan sulfate proteoglycan, might induce maturation of dendritic cells without exogenous stimuli, thus contributing to the generation and maintenance of primary immune responses.

Regulation of T cell homeostasis by heparan sulfate-bound IL-2.

Although IL-2 is commonly thought to promote proliferation of T lymphocytes, mice deficient in IL-2 exhibit splenomegaly, lymphocytosis, and autoimmunity, suggesting this cytokine may have a prominent role in T cell homeostasis. Since the number of T cells in the bloodstream and lymphoid organs is tightly controlled, it is likely that the availability of IL-2 must also be closely regulated. One mechanism altering the local availability of cytokines is association with heparan sulfate, a glycosaminoglycan found on cell surfaces and within extracellular matrices. Here we show that an association between IL-2 and heparan sulfate localizes IL-2 to lymphoid organs such as the spleen. We also show that IL-2, sequestered in this way, contributes to the activation of T lymphocytes and primes T lymphocytes for activation-induced cell death.

Modulation of macrophage and B cell function by glycosaminoglycans.

There is increasing evidence that the behavior of antigen-presenting cells may be regulated, in part, by the surrounding microenvironment. Components of the microenvironment of solid tissues that might influence antigen-presenting cell functions include glycosaminoglycans. We previously showed that heparan sulfate glycosaminoglycans activate macrophages, leading to profound alterations in T cell responses. Here we demonstrate the functional changes that occur in murine antigen-presenting cells induced by heparan sulfate and other glycosaminoglycans, and postulate how these functional changes influence the nature of local immune responses. Heparan sulfate triggered up-regulation of ICAM-1 and I-A, caused the release by antigen-presenting cells of interleukin (IL)-1, IL-6, tumor necrosis factor, IL-12, transforming growth factor beta, and prostaglandin E2 (PGE2), and (in macrophages) induced cytotoxic capability. Heparin induced IL-12 and interferon-gamma production but did not promote the release of other cytokines. Chondroitin sulfate and dermatan sulfate, although not stimulating the production of cytokines or of PGE2, elicited the production by macrophages of nitric oxide. These findings support a model in which the glycosaminoglycan composition of a given tissue, which may be altered by inflammatory processes, helps to regulate the behavior of antigen-presenting cells, which in turn determines the characteristics of the immune response that ensues.

Long-term quality of life after kidney and simultaneous pancreas-kidney transplantation.

We are using a validated questionnaire (SF-36) to annually assess health-related quality of life (QOL) in kidney and pancreas-kidney transplant recipients. The SF-36 consists of eight scales to assess physical functioning, general health, and mental functioning. Norms and 95% confidence intervals (C.I.) have been developed for the US population. At present, 1138 recipients with functioning grafts (520 Type I diabetic; 618 nondiabetic) 1-10 yr post-transplant have completed the questionnaire. Of the recipients, 446 completed the questionnaire once; 632 twice; and 53 three times (305 after 1 yr; 266 after 2 yr; 256 after 3 yr; 206 after 4 yr; 192 after 5 yr; 150 after 6 yr; 130 after 7 yr; 138 after 8 yr; 125 after 9 yr; 92 after 10 yr). For both diabetic and nondiabetic recipients, there was little change in average scores for each scale between years (p = NS). In relation to the US population, average scores for nondiabetics were below the 50th percentile on all 8 scales; for diabetics < 25th percentile on the physical functioning and vitality scales, < 50th percentile on all others. For both diabetic and nondiabetic recipients, average scores were higher than reported norms for patients with CHF, COPD, or depression but were similar to those with Htn or recent MI. Individual scores were then compared with age-matched means (+/- 2 SEMs) (95% C.I.) for the US population. At each year post-transplant, up to 40% of nondiabetic and up to 65% of diabetic recipients had scores below the 95% C.I. on individual scales (particularly the physical functioning and general health scales)--e.g. over 30% nondiabetic and up to 60% diabetic recipients had scores on the physical functioning scales below the 95% C.I. More diabetic recipients (vs. nondiabetics) reported poor QOL on the physical functioning, general health and social functioning scales. There was little difference in the mental health scales. For those with Type I diabetes, a similar percentage of kidney and K/P recipients reported QOL below the 95% C.I. for the age-matched population, except on the GH scale (better QOL for K/P recipients). We conclude that successful transplant recipients report health-related QOL below that of the age-matched general population but similar to those with other chronic diseases. Diabetic and nondiabetic recipients have similar scores on the mental health scales; nondiabetic recipients score better on the general health and physical functioning scales.

The acutely ischemic extremity after kidney transplant: an approach to management.

BACKGROUND: The purpose of this study was to review arterial thromboembolic complications presenting with an acutely ischemic lower extremity after a kidney (KTx) or simultaneous kidney-pancreas transplantation (SPK) and to describe an approach to their management. METHODS: We retrospectively reviewed all such transplantations (a total of 2109) performed between January 1985 and August 1995. We identified 16 recipients (incidence, 0.76%) in whom an acutely ischemic leg developed during the immediate postoperative period (within the first 48 hours). RESULTS: Of the 16 recipients, eight underwent a KTx (incidence, 0.45%) and eight underwent an SPK transplantation (incidence, 2.90%). Median age was 38 years (range, 15 months to 61 years). Thirteen had insulin-dependent diabetes mellitus (IDDM), a significantly higher incidence than in the control group (i.e., transplant recipients without this complication) (p < 0.01). Peripheral vascular disease (PVD) was documented before operation in eight (50%) of the recipients (vs 8.9% in the control group) (p < 0.01). Ten were uremic (on chronic dialysis) before transplantation; six were nonuremic (not on dialysis). Intraoperatively, 14 had moderate to severe atherosclerotic disease affecting the iliac vessels, seven of whom required some manipulation of the artery (either endarterectomy or tacking of the intima) to make it suitable for anastomosis. Heparin was administered systemically during cross clamping to only four. Most of the 16 recipients showed symptoms or signs of arterial occlusion within the first few hours after operation. The most common symptom was pain; the most common physical finding was loss of femoral and distal pulses. Thirteen recipients had moderate to severe ischemia, as judged by physical examination; 15 returned to the operating room for surgical exploration. Eight underwent thrombectomy through an inguinal incision, with successful restoration of flow. Seven underwent exploration through the initial incision because of concern regarding the viability of the transplanted organ; five of them required transplant nephrectomy because of simultaneous thrombosis of the renal artery. No patient needed a bypass procedure to restore flow. Long-term morbidity as a result of the arterial occlusion was related to the severity and length of ischemia. CONCLUSIONS: On the basis of these results, we suggest the following recommendations: (1) all patients should undergo a thorough peripheral vascular examination before and after transplantation; (2) patients at higher risk for arterial thromboembolic complications (e.g., those with significantly diseased vessel at intraoperative examination, nonuremic patients) should receive intraoperative systemic heparin before cross clamping of the artery; and (3) patients with signs or symptoms suggesting arterial occlusion after operation should undergo prompt surgical exploration.

Venous thromboembolic complications after kidney and kidney-pancreas transplantation: a multivariate analysis.

BACKGROUND: We reviewed the incidence of and risk factors for venous thromboembolic complications in our population of kidney (KTx) and simultaneous kidney-pancreas transplant (SPK) recipients. METHODS: Information was collected retrospectively from a database on 1833 KTx and 276 SPK recipients who underwent transplant surgery between January 1985 and August 1995. RESULTS: The incidence of deep venous thrombosis (DVT) was 6.2% (n= 132), with significantly higher rates after SPK (18.1%) vs. KTx (4.5%) (P < 0.001). The number of DVT episodes was highest in the first month; 17.5% occurred during this time. For KTx recipients, early thrombotic events were more common on the side of the graft (P=0.03); however, after 1 month, no correlation existed between the side of the graft and the side of DVT. For SPK recipients, DVT tended to be more common on the side of the pancreas (57%) vs. the kidney (43%) (P=0.10). By multivariate analysis, risk factors for DVT were: age > 40 years (odds ratio [OR]=2.2, P < 0.001), diabetes mellitus (DM) (OR=2.0, P=0.002), previous DVT (OR=4.4, P=0.001), and SPK transplant (OR=2.8, P < 0.001). Pulmonary embolus (PE) was identified in 44 recipients (incidence, 2.1%) and was fatal in 13 (30%). The incidence was significantly higher in SPK (4.71%) vs. KTx recipients (1.69%) (P < 0.01). The risk of death from PE was 0.5% in KTx recipients and 1.37% in SPK recipients (P=0.08). Risk factors for PE included DM (OR=2.6, P=0.005) and recent DVT (OR=8.9, P=0.0001). CONCLUSIONS: Based on risk and extrapolating from the general surgical literature, our recommendations for prophylaxis against DVT are use of graduated compression stockings for all recipients and, in addition, low-dose heparin for moderate and high-risk recipients (previous DVT, SPK, age > 40 years, DM).

Pregnancy after donor nephrectomy.

Potential female donors frequently ask whether unilateral nephrectomy will impair future childbearing capabilities. To address this question, we surveyed 220 women who underwent donor nephrectomy between 1985 and 1992. Of the 144 women who responded, 33 became pregnant after donation for a total of 45 pregnancies. Seventy-five percent of the pregnancies were carried to term without difficulty. Complications incurred during gestation included miscarriage (13.3%), preeclampsia (4.4%), gestational hypertension (4.4%), proteinuria (4.4%), and tubal pregnancy (2.2%). Four of the 45 pregnancies (excluding miscarriages) required preterm hospitalization, resulting in an overall morbidity of 8.8%. There were no pregnancy-related deaths, and no fetal abnormalities were reported. Problems with persistent hypertension, proteinuria, or changes in renal function were not noted. None of the above complications exceeded what has been noted for the general population. Infertility was a problem in 8.3% (3/36) of our respondents, compared with a worldwide incidence of 16.7%. Based on these results, we conclude that donor nephrectomy is not detrimental to the prenatal course or outcome of future pregnancies.

Heparan sulfate initiates signals in murine macrophages leading to divergent biologic outcomes.

We have previously shown that the interaction of heparan sulfate (HS), a constituent of cell surfaces and extracellular matrices, with murine macrophages causes activation of the macrophages leading to the production of cytokines and PGE2 and profound changes in the cellular immune responses triggered by the macrophages. Here we describe the molecular mechanisms that underlie these immunoregulatory changes. We demonstrate that HS delivers signals to macrophages through at least two pathways, one involving the activation of a tyrosine kinase and of nuclear factor-KB, and the other involving the activation of protein kinase C and the elevation of intracellular calcium. The former pathway is associated with the production of IL-6, and the latter pathway is associated with the production of PGE2. Our findings suggest a model in which components of the microenvironment, such as HS, may determine the functional state of an APC, thereby modifying immune responses.

Modulation of cytolytic T cell responses by heparan sulfate.

Accumulating evidence suggests that the functional properties of alloactivated T cells may depend upon the microenvironment in which the T cells reside. For instance, we showed previously that heparan sulfate, a biologically active polysaccharide present on cell surfaces and extracellular matrices, modulates the proliferative responses of splenocytes through enhancement of cytokine and prostaglandin production by macrophages. Here we report that under conditions of suboptimal stimulation, heparan sulfate causes discrete alterations in the functional responses of murine cytolytic T cells. When present in a 5-day mixed leukocyte culture (MLC), heparan sulfate mediates an increase, from 3- to 10-fold, in T cell-mediated cytotoxicity. This increase is dose dependent and most pronounced when heparan sulfate is present in the highest concentration during the first 24 hr of the culture period. On the other hand, when added during the last 48-72 hr of an MLC, heparan sulfate decreases cytotoxicity by 3- to 30-fold. Neutralizing antibodies against IL-1 alpha, but not antibodies against IL1 beta, IL-6, or TNF alpha/beta, abrogate the heparan sulfate-mediated increase in cytotoxicity, suggesting that the increase depended in part upon the production of IL-1 alpha. However, studies in which exogenous IL-1 was added to MLC showed that increased cytotoxicity was not due only to increased cytokine production. Augmentation of cytotoxicity was in part independent of T cell help, as depletion of CD4+ cells from the responder population before MLC, or addition of neutralizing anti-murine IL-2 antibodies plus human IL-2 to the MLC, did not abrogate the stimulatory effect of heparan sulfate. Heparan sulfate-treated CD8+ lymphoblasts isolated after 7 days in MLC demonstrated an increased cytotoxicity, elevated intracellular serine esterase, and perforin levels compared with lymphoblasts from control MLC. The decrease in cytotoxicity observed when heparan sulfate was present during the last several days of an MLC was likely mediated by PGE2, as elevated levels of PGE2 were detected in MLC supernatants of heparan sulfate-treated cultures, and because the decrease was not observed in the presence of indomethacin. Our results are consistent with the idea that the metabolism of heparan sulfate, an endogenous component of parenchymal tissues, may regulate the tempo and magnitude of alloreactive cytotoxic T cell responses.

Regulation by heparan sulfate and interleukin 1 alpha of the ontogenic expression of T-cell receptor, CD4, and CD8 in developing thymus.

Increasing expression by T-cell precursors of the T-cell antigen receptor and of the CD4 and CD8 glycoproteins during thymus development enables specific interactions between primitive T cells and thymic epithelial cells, which initiate the development of the T-cell repertoire. Given the importance of GAGs, such as HS, in cell-cell interactions in other organs, we asked whether such molecules might participate in the cellular interactions essential for the development of the thymus. Using an organ culture model in which fetal murine thymuses explanted on gestational day 14 undergo phenotypic maturation from CD3-CD4-CD8- to CD3+CD4+/CD8+, the consequences of inhibiting GAG synthesis with 6-diazo-5-oxo-L-norleucine (DON) were explored. Inhibition of GAG synthesis prevented elaboration of cortical thymocyte-associated HS, IL-1, and de novo expression of the TCR, CD4, and CD8. DON did not alter the expression of TCR or CD4/CD8 by mature thymocytes. Synthesis of IL-1 alpha, TCR, CD4, and CD8 was restored by addition of HS to the cultured organs. These findings support the concept that the ontogenic (but not the constitutive) expression of the TCR and of CD4 and CD8 depends on the synthesis in the thymus of IL-1, and that IL-1 synthesis is in turn regulated by the metabolism of GAGs. Our findings suggest that components of the thymic microenvironment, particularly HS, play a critical role in the maturation of T-cell precursors.

Role of heparan sulfate in immune system-blood vessel interactions.

Heparan sulfate proteoglycan, a component of endothelial cell membranes and extracellular matrices, is involved in a number of the critical functions of endothelium and of antigen-presenting cells. This review discusses how heparan sulfate is released from endothelial cells during inflammation, how the loss of heparan sulfate potentially alters endothelial cell physiology, and how the presence of heparan sulfate in a soluble form might regulate the functioning of lymphocytes at sites of inflammation.

Regulation of murine splenocyte responses by heparan sulfate.

Heparan sulfate is a biologically active glycosaminoglycan found in abundance in endothelium, epithelium, and connective tissues. Although heparan sulfate is an important part of the environment in which immune cells function, its effects on the immune system are largely unknown. When present during the first 24 h of an MLC consisting of MHC disparate murine splenocytes, heparan sulfate had a marked stimulatory effect on the proliferative response to alloantigens. Heparan sulfate also augmented the splenocyte response to suboptimal concentrations of the mitogens Con A, anti-CD3 mAb, and ionomycin. The stimulatory action of heparan sulfate was mediated, at least in part, by increased production of IL-1, because the increased splenocyte proliferation induced by heparan sulfate was substantially inhibited by anti-murine IL-1 alpha-antibodies. In contrast to these stimulatory effects, when heparan sulfate was added to MLC 48 to 72 h after onset, decreased splenocyte proliferation was observed. This inhibitory action was mediated by an increase in PGE2 production; the inhibitory effect could be abrogated with indomethacin. The fact that heparan sulfate is present on cells such as endothelial cells with which T cells interact and is released during activation of endothelial cells (thus making it available in soluble form to cells in the immune response) may allow heparan sulfate to play an important role in modulating cell-mediated immune responses in vivo.