Mark D. Denton

CD40 and CD40 ligand (CD154) are coexpressed on microvessels in vivo in human cardiac allograft rejection

BACKGROUND CD40 is expressed by a wide variety of cells in the immune system, including endothelial cells. It binds to CD40 ligand ([CD40L] CD154), which was originally reported to be restricted in its expression to early-activated T cells. We report here the expression of CD40 and CD40L in human cardiac allografts. METHODS A total of 123 consecutive biopsies from 11 human cardiac allograft recipients were analyzed by immunohistochemistry for the expression of CD40 and CD40L. The expression of CD40L was also examined in vitro in homogeneous cultures of umbilical vein endothelial cells by reverse transcriptase-polymerase chain reaction and by flow cytometry. RESULTS CD40 was expressed at low levels, and CD40L was minimal or absent in histologically normal biopsies in the absence of CD3+ T-cell infiltrates. In rejection, the expression of CD40 increased on vascular endothelial cells and on graft-infiltrating leukocytes throughout biopsy specimens. Induced expression of CD40 was strongly associated with the presence of CD3+ T-cell infiltrates, acute rejection, and ischemic injury (P<0.05). CD40L was expressed in biopsies with rejection and was prominent on a subset of infiltrating leukocytes as well as on microvascular endothelial cells. In contrast to CD40, staining of endothelial CD40L was focal in most biopsies. Overall, the expression of CD40L correlated with the presence of CD3+ T-cell infiltrates and rejection (P<0.05), but not ischemic injury (P=0.9). To confirm that the endothelium can synthesize CD40L, we also evaluated the expression of endothelial CD40L in vitro. Cultured endothelial cells were found to express little constitutive CD40L that markedly increased after 24 hr of treatment with supernatants from phytohemagglutinin-activated peripheral blood mononuclear cells or by the cytokines tumor necrosis factor-alpha, interleukin-1a, interleukin-4, or interferon-gamma. CONCLUSION Both CD40 and CD40L are expressed in vivo on infiltrating leukocytes and on microvascular endothelium in human cardiac allograft rejection. We suggest that endothelial cell CD40 and CD40L play a role in human cell-mediated immune responses such as cardiac allograft rejection.

The role of the graft endothelium in transplant rejection: Evidence that endothelial activation may serve as a clinical marker for the development of chronic rejection

Abstract: In this review, we discuss the role of the allograft endothelium in the recruitment and activation of leukocytes during acute and chronic rejection. We discuss associations among endothelial activation responses, the expression of adhesion molecules, chemokines and chemokine receptors, and rejection; and we propose that endothelial vascular cellular adhesion molecule‐1 (VCAM‐1) may be used as a surrogate marker of acute rejection and allograft vasculopathy. In addition, we describe potential mechanistic interpretations of persistent endothelial cell (EC) expression of major histocompatibility complex (MHC) class II molecules in allorecognition. The graft endothelium may provide an antigen‐specific signal to transmigrating, previously activated, T cells and may induce B7 expression on locally transmigrating leukocytes to promote costimulation. Taken together, these functions of the EC provide it with a potent regulatory role in rejection and in the maintenance of T‐cell activation via the direct and/or the indirect pathways of allorecognition.

Sirolimus‐Induced Pneumonitis: Three Cases and a Review of the Literature

Interstitial pneumonitis is a rare disease that is seen in the context of some infections (e.g. PCP and CMV pneumonia), as side‐effects of drugs (e.g. beta‐blockers, amiodarone) and rarely in the context of renal transplantation. It manifests itself usually as a pneumonic illness; with symptoms of dyspnea, cough, fatigue and sometimes fever. Characteristic radiological changes are bilateral lower zone haziness. Interstitial pneumonitis is now emerging in solid organ transplant patients secondary to sirolimus). We describe three cases of sirolimus‐induced pneumonitis in two patients who started sirolimus to permit cyclosporin withdrawal and in one patient initially started on sirolimus. The presentations in these cases ranged from insidious to fulminant; there was a rapid response to sirolimus withdrawal. This is an important syndrome, with an unknown frequency.

CD28-B7-Mediated T Cell Costimulation in Chronic Cardiac Allograft Rejection : Differential Role of B7–1 in Initiation versus Progression of Graft Arteriosclerosis

Provision of adequate T cell costimulation is critical for the development of acute and chronic allograft rejection. We have previously reported that early blockade of CD28-B7 T cell costimulation prevents the development of graft arteriosclerosis, in the LEW into F344 rat cardiac transplant model. In this study, we used the same model to examine the requirement for CD28-B7-mediated T cell costimulation in the progression of established chronic rejection and examined the individual roles of B7-1 (CD80) and B7-2 (CD86) costimulatory molecules. Late blockade of CD28-B7 T cell costimulation by the fusion protein CTLA4Ig, which binds both CD80 and CD86, attenuated the development of transplant arteriosclerosis, mononuclear cell infiltration, and parenchymal fibrosis in this model. Selective blockade of CD80 using the mutant fusion protein Y100F was as effective as CTLA4Ig in this regard. In contrast to CTLA4Ig, blockade of CD80 alone by Y100F was ineffective at preventing early graft loss and prolonging graft survival when given early after transplantation. This study is the first to demonstrate that late blockade of CD28-B7 T cell costimulation interrupts chronic cardiac allograft rejection, and it indicates the importance of continued T cell activation in this process. This study further defines functional differences between CD80 and CD86 costimulatory molecules in vivo.

Specific MDR1 P-Glycoprotein Blockade Inhibits Human Alloimmune T Cell Activation In Vitro

MDR1 P-glycoprotein (P-gp), the multidrug resistance-associated transmembrane transporter, is physiologically expressed by human peripheral immune cells, but its role in cell-mediated immunity remains poorly understood. Here, we demonstrate a novel role for P-gp in alloantigen-dependent human T cell activation. The pharmacologic P-gp inhibitor tamoxifen (1–10 μM) and the MDR1 P-gp-specific mAb Hyb-241 (1–20 μg/ml), which detected surface P-gp on 21% of human CD3+ T cells and 84% of CD14+ APCs in our studies, inhibited alloantigen-dependent, but not mitogen-dependent, T cell proliferation in a dose-dependent manner from 40–90% (p < 0.01). The specific inhibitory effect on alloimmune T cell activation was associated with >85% inhibition (p < 0.01) of IL-2, IFN-γ, and TNF-α production in 48-h MLR coculture supernatants. Addition of recombinant human IL-2 (0.1–10 ng/ml) restored proliferation in tamoxifen-treated cocultures. Pretreatment of purified CD4+ T cells with Hyb-241 mAb before coculture resulted in inhibition of CD4+ T cellular IFN-γ secretion. Also, blockade of P-gp on allogeneic APCs inhibited IL-12 secretion. Taken together these results demonstrate that P-gp is functional on both CD4+ T cells and CD14+ APCs, and that P-gp blockade may attenuate both IFN-γ and IL-12 through a positive feedback loop. Our results define a novel role for P-gp in alloimmunity and thus raise the intriguing possibility that P-gp may represent a novel therapeutic target in allograft rejection.

Significance of Anticardiolipin Antibodies on Short and Long Term Allograft Survival and Function following Kidney Transplantation

The significance of anticardiolipin antibodies (ACAs) prior to renal transplantation is unclear. We studied a cohort of 337 patients who underwent renal transplantation from 1996 to 2001. Follow‐up continued until allograft loss, patient death or 31 December 2002. The primary outcome was a composite endpoint of death‐censored allograft loss or a 25% reduction in estimated glomerular filtration rate (GFR) from 1‐month post‐transplant. Secondary outcomes were allograft loss, a 25% reduction in GFR, acute rejection and creatinine at 1 year. IgG and IgM ACA titers were positive (≥15) in 18.1% of recipients. There were no significant differences at baseline between recipients, except coumadin therapy in those with positive ACA titers (20% vs. 7.4%). Post‐transplant, there was no increase in the primary outcome in ACA‐positive patients, even after adjustment for anticoagulation with coumadin (HR = 1.42 [0.68, 2.96]). There was no difference in secondary outcomes between those with or without positive titers. Two of five patients with very high titers (>50) who were not anticoagulated had early graft loss. A positive ACA titer prior to kidney transplantation was not associated with inferior renal outcomes after transplantation, although more research is required to address the prognostic significance of very high ACA titers.

Membranous Lupus Nephritis in a Renal Allograft: Response to Mycophenolate Mofetil Therapy

Membranous lupus nephritis in a renal allograft is considered rare. A 43‐year‐old man with quiescent systemic lupus erythematosus (SLE) received a HLA identical transplant from his sister and 4 years later developed persistent nephrotic range proteinuria and morphological features most compatible with membranous lupus nephritis on biopsy. Angiotensin‐converting enzyme (ACE) inhibitors and angiotensin II receptor antagonists, although successful in reducing proteinuria, were associated on three occasions with acute allograft dysfunction. Sustained reduction of proteinuria and stable graft function were achieved using mycophenolate mofetil (MMF). MMF is emerging as a new therapy for primary renal disease in SLE. This is the first report of successful treatment of membranous lupus nephritis in an allograft using MMF. We review all cases of transplant‐associated membranous lupus nephritis in the English literature.

Immunomodulatory functions of hyaluronate in the LEW-to-F344 model of chronic cardiac allograft rejection.

BACKGROUND CD44 is an important leukocyte cell surface glycoprotein with diverse functions including cell adhesion, homing, migration, and activation. METHODS Because administration of the principal ligand of CD44, hyaluronate (HA), in soluble form, can inhibit CD44-HA interaction, we tested the effects of HA in vivo in an established model of chronic allograft rejection. Control F344 recipients of LEW hearts received either no treatment or low-dose cyclosporine (CsA) for 30 days from the day of transplantation. Experimental animals received 30 days of CsA in combination with 30 or 90 days of low molecular weight HA (LMW-HA). RESULTS CsA therapy alone resulted in approximately 40% long-term (>100 days) graft survival, whereas CsA + LMW-HA (30-day and 90-day protocols) significantly increased long-term graft survival to 60% and 92%, respectively. Light microscopy and immunohistology of CsA-treated and CsA + LMW-HA-treated grafts harvested at day 30 after transplantation demonstrated that LMW-HA + CsA therapy decreased mononuclear cell infiltration and afforded better preservation of myocardial architecture. In addition, LMW-HA + CsA-treated grafts exhibited decreased expression of interferon-gamma and the growth factors transforming growth factor-beta, platelet-derived growth factor, and fibrogenic growth factor-beta. Long-term surviving grafts were assessed for arteriosclerosis, the sine qua non of chronic rejection in this model. Using a standardized scoring system, significantly less arteriosclerosis was seen in grafts from LMW-HA + CsA-treated animals at 120 days after transplantation compared with CsA alone-treated grafts. This difference was not significant, however, in grafts harvested at >150 days. CONCLUSION This is the first report indicating that CD44-HA interactions play an important role in chronic allograft rejection.

Early results of a controlled non-heart-beating kidney donor programme

BACKGROUND We present our experience of a controlled non-heart beating donation (CNHBD) programme in a University Hospital. METHODS Data from all referrals for CNHBD between January 2005 and January 2008 were collected prospectively. Donor and recipient data were analysed and compared to other cadaveric and HBD transplants performed during the same period. RESULTS During the period, 79 donors were referred resulting in 35 proceeding to retrieval and 61 kidneys being successfully transplanted. The median time from withdrawal of therapy to asystole was 15 min (IQR 10.0-23.0). The median primary warm ischaemic time was 20 min (IQR 16.0-27.0). The mean cold ischaemia time was 16.6 +/- 4.21 h for CNHBD (16.6 +/- 5.91 for HBD) kidneys. Compared to HBD kidneys, CNHBD kidneys had more HLA mismatches and significantly more delayed graft function (44% versus 14%), and the mean time to halving of serum creatinine was significantly greater (12.8 versus 5 days). However, 1-year patient and graft survival (88% and 93%) were excellent and mean creatinine at 12 months for CNHB kidneys was not significantly different from HBD kidneys (141 mumol/l versus 131 mumol/l). CONCLUSIONS Structured implementation resulted in a successful CNHBD programme providing 61 successful renal transplants from 35 donors in 3 years-contributing to approximately 50% of the total number of cadaveric renal transplants during the period. At 12 months, CNHBD kidney graft function was equivalent to HBD organs.

Assessment by flow cytometry of intracellular cytokine production in the peripheral blood cells of renal transplant recipients

Abstract:  Introduction:  There is accumulating evidence that non‐invasive immune monitoring may be useful in the early period after renal transplant, particularly with regard to predicting the presence of acute rejection. It is less clear whether chronic allograft nephropathy (CAN) is also associated with consistent changes in peripheral blood or urine cells. We hypothesized that patients with CAN would manifest different patterns of cytokine production (compared with non‐CAN controls), detectable in peripheral blood mononuclear cells (PBMCs).