Multicenter Analysis of Immune Biomarkers and Heart Transplant Outcomes: Results of the Clinical Trials in Organ Transplantation-05 Study.

Identification of biomarkers that assess posttransplant risk is needed to improve long-term outcomes following heart transplantation. The Clinical Trials in Organ Transplantation (CTOT)-05 protocol was an observational, multicenter, cohort study of 200 heart transplant recipients followed for the first posttransplant year. The primary endpoint was a composite of death, graft loss/retransplantation, biopsy-proven acute rejection (BPAR), and cardiac allograft vasculopathy (CAV) as defined by intravascular ultrasound (IVUS). We serially measured anti-HLA- and auto-antibodies, angiogenic proteins, peripheral blood allo-reactivity, and peripheral blood gene expression patterns. We correlated assay results and clinical characteristics with the composite endpoint and its components. The composite endpoint was associated with older donor allografts (p < 0.03) and with recipient anti-HLA antibody (p < 0.04). Recipient CMV-negativity (regardless of donor status) was associated with BPAR (p < 0.001), and increases in plasma vascular endothelial growth factor-C (OR 20; 95%CI:1.9-218) combined with decreases in endothelin-1 (OR 0.14; 95%CI:0.02-0.97) associated with CAV. The remaining biomarkers showed no relationships with the study endpoints. While suboptimal endpoint definitions and lower than anticipated event rates were identified as potential study limitations, the results of this multicenter study do not yet support routine use of the selected assays as noninvasive approaches to detect BPAR and/or CAV following heart transplantation.

mTOR-understanding the clinical effects.

The target of rapamycin (TOR) is a highly conserved serine/threonine kinase that controls cell growth and metabolism in response to nutrients, growth factors, cellular energy, and stress. The TOR kinase, which was originally discovered in yeast, is also expressed in human cells as mammalian TOR (mTOR). In this review, we focus on how mTOR-inducible signals function in cell protection and cell survival of effector and regulatory T cells as well as its role in endothelial cell biology. We evaluate how signaling is important for vascular endothelial cell growth, survival, and proliferation; and we consider how the function of mTOR in endothelial cells may be clinically important in the rejection process. Understanding the biology of mTOR allows clinicians to use mTOR inhibitors optimally as therapeutics following solid organ transplantation.

Expression of the chemokine receptor CXCR3 and its ligand IP-10 during human cardiac allograft rejection.

BACKGROUND: Chemokines play an essential role in regulating the infiltration of leukocytes into allografts in experimental models. Little is known of their expression or function after human cardiac transplantation. METHODS AND RESULTS: We analyzed 169 sequential human endomyocardial biopsies by immunocytochemistry for infiltration by CD3(+) T cells and the expression of the chemokine receptors CCR1, CCR3, CCR5, and CXCR3. In both cross-sectional and longitudinal analyses, the expression of each of the chemokine receptors correlated with the degree of CD3(+) T-cell infiltration. In particular, the expression of CXCR3 was temporally and spatially associated with CD3(+) T-cell infiltrates and correlated with the histopathological diagnosis of acute rejection (OR, 11.73 and 4.05, respectively; P<0.001). Of 7 patients followed up longitudinally for 1 year, 4 with consecutive biopsies developed intimal thickening by intravascular ultrasound. In these patients, there was a trend for persistent expression of CD3- and CXCR3-expressing infiltrates in the later part of the first posttransplant year. The chemokines eotaxin, IP-10, lymphotactin, MCP-1, Mig, RANTES, and SDF-1 were examined in an additional 35 biopsies by RT-PCR. Eotaxin, lymphotactin, MCP-1, Mig, and SDF-1 were present in both normal and rejecting biopsies. However, the CXCR3 ligand IP-10, which was rarely expressed in normal biopsies, was markedly induced in acute rejection (OR, 19.43; P=0.01). CONCLUSIONS: The presence of CXCR3(+) T cells and the CXCR3 ligand IP-10 within endomyocardial biopsies is strongly associated with acute rejection. The CXCR3-IP-10 interaction warrants consideration as a therapeutic target in the management of cardiac allograft recipients.

Risk factors for mortality in infants and young children on dialysis.

The factors associated with a greater mortality risk in infants and young children undergoing dialysis have not been clearly determined. We report the results of a North American Pediatric Renal Transplant Cooperative Study designed to assess risk factors in patients aged younger than 6 years at initiation of dialysis therapy. Sixty-four nonsurvivors were matched with 110 survivors for age at dialysis initiation, primary renal disease, and year of entry onto the database. Questionnaires on 137 patients (51 nonsurvivors, 86 survivors) were completed by participating centers. Seventy-five percent (103 of 137 patients) of the patients were aged younger than 2 years at dialysis initiation; 42% (58 of 137 patients) had renal aplasia, dysplasia, and/or hypoplasia or obstructive uropathy; 62% were boys; and 62% were white. One-year patient survival rates were 83% in infants beginning dialysis at younger than 3 months of age, 89% in 3- to 23-month-olds, and 95% in 2- to 5-year-olds (P = 0.001). Comorbid nonrenal disease occurred in 37 of 51 nonsurvivors (74%) versus 46 of 84 survivors (55%; P = 0.027). Nonsurvivors had pulmonary disease and/or hypoplasia more often (14 of 37 nonsurvivors; 37.8% versus 8 of 46 survivors; 17.4%; P = 0.04). Oliguria or anuria was present in 23 of 33 nonsurvivors (70%) aged younger than 2 years versus 26 of 64 survivors (41%; P = 0.007). Infection accounted for 15 of 51 deaths (29.4%). In summary, these results suggest that age at dialysis initiation; presence of nonrenal disease, particularly pulmonary disease and/or hypoplasia; and oliguria or anuria in children aged younger than 2 years are identifiable as risk factors for mortality in these young patients.

Ligation of CD40 induces the expression of vascular endothelial growth factor by endothelial cells and monocytes and promotes angiogenesis in vivo.

This study addresses a mechanism by which lymphocytes may promote vascular endothelial growth factor (VEGF) expression and angiogenesis in immune inflammation. Resting human umbilical endothelial cells (HUVECs) were found to express low levels of VEGF messenger RNA (mRNA) by reverse transcription polymerase chain reaction and ribonuclease protection assay with little or no change in expression following activation by cytokines, including tumor necrosis factor-alpha, interleukin (IL)-1, interferon gamma, or IL-4. In contrast, treatment of HUVECs and monocytes with soluble CD40 ligand (sCD40L) resulted in a marked dose-dependent induction of VEGF mRNA (approximately 4-fold), which peaked between 1 and 5 hours post-stimulation. Transient transfection of HUVECs was performed with a luciferase reporter construct under the control of the human VEGF promoter. Treatment of transfected HUVECs with sCD40L was found to enhance luciferase activity (approximately 4-fold) compared with controls, similar to the relative fold induction in mRNA expression in parallel cultures. Thus, CD40-dependent VEGF expression was a result of transcriptional control mechanisms. Treatment of HUVECs with sCD40L was also found to function in vitro to promote growth and proliferation in a VEGF-dependent manner, and CD40-dependent HUVEC growth was comparable to that found following treatment with recombinant human VEGF. Furthermore, subcutaneous injection of sCD40L in severe combined immunodeficient and nude mice induced VEGF expression and marked angiogenesis in vivo. Taken together, these findings are consistent with a function for CD40L-CD40 interactions in VEGF-induced angiogenesis and define a mechanistic link between the immune response and angiogenesis. (Blood. 2000;96:3801-3808)

Interactions between T lymphocytes and endothelial cells in allograft rejection.

Vascular endothelial cells participate in the process of allograft rejection by promoting both the recruitment and the activation of alloreactive T cells. There have been three major recent advances in the field of interactions between T cells and endothelial cells that are of direct relevance to the process of cell-mediated responses to allografts: first, endothelial cells mediate selective recruitment of CD4+ T cell subsets, including naive and memory T cells and T cell subsets of the Th1 and Th2 phenotypes; second, endothelial cells co-stimulate the production of effector cytokines by helper T cells; and third, endothelial cells regulate T cell apoptosis.

Central role for CD40/CD40 ligand (CD154) interactions in transplant rejection.

Major advances have been made in understanding the expression and function of CD40 and its ligand CD154. It is now clear that CD40/CD154 interactions are critical in many aspects of the immune response, including T cell activation, T cell-dependent macrophage activation, T cell-B cell interactions and endothelial activation. Moreover, increasing evidence supports a central role for CD40/CD154 interactions in the immune processes of allograft rejection. Functional studies using blocking monoclonal antibodies have revealed beneficial effects of interupting CD40/CD154 co-stimulation in animal models of transplantation, particularly in association with interuption of the CD28/B7 pathway. A next step is to develop new therapeutic approaches to interrupting this pathway in humans, either through the development of receptor antagonists or through the understanding of intracellular signaling pathways utilized by these molecules.

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.

P-selectin expression in myocardium of children undergoing cardiopulmonary bypass.

Cardiopulmonary bypass is a planned support technique that results in a period of myocardial ischemia and reperfusion. In addition, it is associated with an inflammatory response likely involving endothelial cell activation. In previous studies, we showed that E-selectin and intercellular adhesion molecule-1 (ICAM-1) messenger ribonucleic acid (mRNA) are increased in human myocardium after cardiopulmonary bypass. We have now examined the expression of P-selectin mRNA by ribonuclease protection in paired atrial biopsy specimens from 12 patients before and after cardiopulmonary bypass. By means of immunocytochemistry, we have also examined the endothelial cell surface expression of P-selectin protein, as well as that of E-selectin and ICAM-1 in three additional patients. Patient ages ranged from 1 day to 8.5 years (median 12 months), and cardiopulmonary bypass times ranged from 46 to 196 minutes (median 144 minutes). By ribonuclease protection, there was marked variability in the expression of P-selectin in biopsy specimens before bypass. However, when compared with prebypass levels, P-selectin mRNA decreased modestly in 10 of 12 patients after bypass (median decrease 1.5-fold, p = 0.016). As seen with immunocytochemistry, P-selectin protein was distributed diffusely through the vascular bed on large vessels and small vessels before bypass but was virtually absent on capillaries in specimens taken after bypass. E-selectin, which was absent in prebypass biopsy specimens, was induced in one of the three specimens after bypass, but no change in ICAM-1 protein expression above baseline was noted. We also find that cultured human endothelial cells treated with tumor necrosis factor-alpha in doses which induce ICAM-1 mRNA simultaneously decrease their expression of P-selectin mRNA as compared with untreated cells. These observations suggest that endothelial P-selectin is transcriptionally downregulated after cardiopulmonary bypass at times when E-selectin and ICAM-1 are induced. Furthermore, we find that E-selectin and ICAM-1 are expressed at times and at sites where P-selectin is absent. Although it is possible that P-selectin may have been induced and lost at early times before reperfusion, these data suggest that endothelial P-selectin plays a limited role in the inflammatory response that ensues after cardiopulmonary bypass.

Predictive value of inducible endothelial cell adhesion molecule expression for acute rejection of human cardiac allografts.

We conducted a prospective longitudinal study to determine the clinical significance of endothelial adhesion molecule expression in endomyocardial biopsies from human cardiac allografts. Ten to 18 (mean 13) consecutive allograft biopsies were obtained from 20 serial human transplant recipients over a one-year period. A total of 267 biopsies was examined. The expression of endothelial adhesion molecules intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin, as well as the presence of CD3+ T cell infiltrates was assessed by immunocytochemical staining of frozen sections. Separate specimens taken at the same time were analyzed histologically for ischemic injury or rejection. ICAM-1--and, to a lesser extent VCAM-1--was expressed at low levels in normal biopsies. E-selectin was only expressed in 15% of histologically normal biopsy specimens. Ischemic injury noted in the immediate posttransplant period was associated with increased expression of all three adhesion molecules. VCAM-1 expression increased both with the degree of CD3+ T cell infiltrates (P < 0.001) and with the degree of rejection (P < 0.05). ICAM-1 increased over constitutive levels in association with diffuse CD3+ infiltrates (P < 0.001) and with rejection (P < 0.05). E-selectin was increased on occasional vessels in association with CD3+ infiltrates (P < 0.001), but was not associated with active rejection. Increases in E-selectin were most likely to occur in biopsies just prior to rejection episodes (odds ratio 3.3), and were least likely to occur in biopsies following rejection (odds ratio 0.3). ICAM-1, but not VCAM-1, was also elevated in prerejection specimens. VCAM-1 and ICAM-1 declined in postrejection specimens. These data suggest a dynamic pattern in the expression of endothelial cell adhesion molecules during the course of cardiac allograft rejection. This study also suggests that endothelial E-selectin expression may be a useful clinical marker of impending rejection. Finally, inducible VCAM-1 expression may be a helpful adjunct in the diagnosis of ongoing acute rejection, and decreases in its expression may be indicative of successful antirejection therapy.

Alpha 1-VIII collagen is expressed in the rat glomerulus and in resident glomerular cells.

Current knowledge regarding the molecular composition of extracellular matrices in the glomerulus does not explain how these components interact to form stable three-dimensional structures. The recent recognition that short-chain collagens such as type VIII collagen function as molecular bridges in some nonrenal tissues has raised the possibility that such molecules may serve a similar function in the glomerulus. We have recently shown that cultured rat mesangial cells synthesize and secrete several short-chain collagenous proteins, one of which has properties similar to alpha 1-VIII collagen. In the present study we have isolated a rat mesangial cell alpha 1-VIII collagen cDNA clone, the sequence of which is 81% homologous to mouse alpha 1-VIII collagen. We used this cDNA to determine that alpha 1-VIII collagen mRNA is expressed in rat renal cortex and in cultured glomerular mesangial, epithelial, and endothelial cells. Additionally, we demonstrated that alpha 1-VIII collagen is secreted by cultured mesangial cells as an 80-kDa translation product. By immunocytochemistry, alpha 1-VIII collagen localized to the media of large intrarenal arteries and to the capillary loops and the mesangium of normal rat kidney. These results indicate that type VIII collagen is a normal constituent of the rat glomerulus as well as large intrarenal arteries. We speculate that type VIII collagen may function in part to determine the three-dimensional organization of the subendothelial and mesangial matrices.

Expression of vascular cell adhesion molecule-1 in human renal allografts.

The expression of vascular cell adhesion molecule-1 (VCAM-1) in 11 human renal allograft biopsies and 3 normal kidney specimens was investigated by immunocytochemistry. VCAM-1 expression was correlated with the degree of CD3+ T cell infiltration and the clinicopathologic diagnosis of acute rejection. CD3+ infiltrates were seen in all biopsies with rejection, but not in normal biopsies or one with acute tubular necrosis, and were accompanied by CD68+ monocyte/macrophage infiltrates. In normal biopsies, VCAM-1 was present on occasional tubules, where its expression was patchy and restricted to the basolateral surface of cells with slight cytoplasmic staining. The total number of tubules expressing VCAM-1 significantly increased in specimens infiltrated with CD3+ T cells. Moreover, in these infiltrated biopsy specimens, VCAM-1 was present throughout the cytoplasm of tubular cells concentrated on the basolateral surface. VCAM-1 was also observed on vascular endothelial cells where its expression correlated with the degree of CD3+ infiltrate. Mean scores (0 to 3+) for endothelial VCAM-1 expression increased from 0 (CD3+ score, 0) to a mean score of 2.25 in association with CD3+ T cell infiltrates (CD3+ score, 3). Endothelial VCAM-1 was predominantly on vessels in areas of infiltrate, including peritubular capillaries, venules, and arterioles, but was notably absent on glomerular endothelium. VCAM-1 also stained mesangial cells in an occasional CD3+ infiltrated specimen. It was concluded that the expression of VCAM-1 is increased on renal tubules and renovascular endothelium in rejecting renal allografts in association with CD3+ infiltrates.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of tumor necrosis factor, lipopolysaccharide, and IL-4 on the expression of vascular cell adhesion molecule-1 in vivo. Correlation with CD3+ T cell infiltration.

We have injected human TNF, LPS, and IL-4 into the skin of baboons to examine regulation of endothelial leukocyte adhesion molecules (ELAM) in vivo and to determine which endothelial adhesion molecules correlate temporally and spatially with cytokine-induced T cell infiltration. The expression of adhesion molecules ELAM-1 (E-selectin), VCAM-1, and ICAM-1 (CD54) were quantified by immunocytochemical staining of frozen sections obtained from skin biopsies; T cell infiltration was measured by immunocytochemical staining of CD3+ T cells in serial sections. We found that injection of TNF causes late (24 to 48 h) T cell infiltration whereas injection of LPS, in doses that do not cause tissue necrosis, does not. The ability of TNF (but not LPS) to recruit T cells correlates with the ability of TNF to cause sustained endothelial cell adhesion molecule expression. Expression of VCAM-1 on post-capillary venules showed the highest degree of spatial localization with infiltrates. IL-4, although not proinflammatory by itself, can cause T cell infiltration in combination with an ineffective dose of TNF. The ability of IL-4 to augment TNF-induced inflammation best correlates with the ability of the combination of IL-4 and TNF to increase endothelial VCAM-1 expression. In contrast, IL-4 does not promote T cell infiltration or endothelial VCAM-1 expression in combination with LPS. In cytokine-injected tissues, VCAM-1 is also expressed on connective tissue cells other than endothelium, including smooth muscle and perineural cells, where it is induced by cytokines in parallel with endothelial VCAM-1. Overall, our data support the hypothesis that endothelial VCAM-1 expression contributes to T cell extravasation at sites of inflammation. Furthermore, we find that IL-4, a product a Ag-activated T cells, can interact with TNF to selectively promote VCAM-1 expression and the development of T cell-rich infiltrates, characteristic of Ag-induced inflammatory reactions.

Childhood membranoproliferative glomerulonephritis type I: limited steroid therapy.

Nineteen patients with biopsy proven membranoproliferative glomerulonephritis type I (MPGN I) and a minimum of three years of follow-up (mean 6.5 +/- 0.7 years) have been treated with an uncontrolled regimen of limited corticosteroids. Initial therapy ranged from 20 mg per os (po) every other day to 30 mg/kg/day i.v. for three consecutive days, depending on clinical disease severity. Therapy was then decreased based on each patient's improving clinical status. At diagnosis creatinine clearance (CCr) was less than 80 ml/min/1.73 m2 in 12 patients and less than 50 in 2. All patients had hematuria and proteinuria, with 15 in the nephrotic range. Hypertension, present at diagnosis in 13, developed in five others following institution of prednisone, and was controlled medically. Renal biopsy was repeated after two years of therapy prior to cessation of treatment (mean total treatment duration 38 +/- 3 months). Follow-up biopsy revealed decreased glomerular inflammatory activity in 88% of patients. All patients have now been off prednisone for 40 +/- 9 months. The mean CCr is 126 +/- 5 ml/min/1.73 m2. Eight patients have normal urinalyses. These data suggest that early therapy with a limited course of corticosteroids, and control of associated hypertension, may forestall progressive renal insufficiency in children with MPGN type I.