Protection of U937 cells against oxidative injury by a novel series of iron chelators

A new series of iron chelators designed to protect tissues against iron-catalysed oxidative damage is described. These compounds are aminocarboxylate derivatives bearing pendant aromatic groups. They were designed to have a relatively low affinity for both ferrous and ferric iron and to be site-specifically oxidizable by hydrogen peroxide through intramolecular aromatic hydroxylation into species with strong iron binding capacity which do not catalyse hydroxyl radical formation. Thus, at the cellular level, oxidative injury is used to convert weak iron chelators into strong iron chelators in order to promote cell survival. The purpose of this local activation process is to minimise toxicity compared to strong iron chelators which may interfere with normal iron metabolism. Compounds within this series were evaluated in vitro in view of their capacity to undergo intramolecular hydroxylation and to protect cultured cells against oxidative injury. Results show that the intramolecular aromatic hydroxylation capacity is critically dependent upon the amino carboxylate chelating moieties and the substituents of the aromatic rings. Cell protection against oxidative injury is only observed with compounds possessing sufficient lipophilicity. The monohydroxylation product of N,N'-dibenzylethylenediamine N,N'-diacetic acid, protects cells against both H2O2 and tBuOOH toxicity with IC50's of 12 and 60 microM, respectively, in agreement with the oxidative activation concept. These results represent the first step toward the development of a new strategy to safe iron chelation for the prevention of oxidative damage.     

mtDNA-depleted U937 cells are sensitive to TNF and Fas-mediated cytotoxicity

It has been proposed that TNF cytotoxicity is mediated by reactive oxygen intermediates generated by uncoupling of mitochondrial respiration. We have compared sensitive U937 cells and derived cell lines depleted of mtDNA for their ability to undergo TNF- and Fas-induced apoptosis. Cells lacking around 98% of mtDNA were still sensitive to TNF-induced apoptosis. U937 cells devoid of mtDNA (U937-rho degree) were resistant to TNF, but this was due to the loss of its 55 kDa receptor. U937-rho degree cells were also resistant to docosahexaenoic acid, which causes U937 cell death by lipid peroxidation. These cells were sensitive to anti-Fas toxicity. The results indicate that TNF and Fas-induced toxicity occurs by a mechanism mostly independent of mitochondrial free radical generation.     

Evidence for binding of in vitro glycated low-density lipoproteins by fructosyllysine-specific sites on macrophages and U937 monocyte-like cells

Early development of arteriosclerosis is a main late complication of diabetes mellitus. Although its uptake by LDL receptors is impaired, glycated LDL is thought to play a role in foam cell formation from macrophages. In the present study we show binding of glycated LDL (8-9 mol fructosyllsine/mol apo B) to macrophages and to the monocyte-like cell line U937. The binding involves fructosyllysine-specific binding sites, as well as LDL and scavenger receptors. Fructosyllysine and glycated albumin were competitors for binding of 125I-labelled glycated LDL by macrophages and U937 cells. Scatchard analysis of binding data using a two ligands binding model showed a linear plot with Ka = 2.6 x 10(7) M-1 for U937 cells, which lack scavenger receptors. On U937 cells only the 200 kDa fructosyllysine-specific receptor protein and the 165 kDa LDL receptor were involved in binding glycated LDL as evidenced by ligand blotting. U937 cell uptake and degradation of glycated LDL was mediated by fructosyllysine-specific and LDL receptors. Binding of glycated LDL by macrophages via fructosyllysine-specific sites could be demonstrated in only 6 from 35 rats investigated, indicating that the receptor is not expressed in each individual. Whether the fructosyllysine-specific receptor mediated pathway is relevant for uptake and degradation of glycated LDL in vivo is yet to be determined.     

Defensin stimulates the binding of lipoprotein (a) to human vascular endothelial and smooth muscle cells

There is evidence to suggest that elevated plasma levels of lipoprotein (a) [Lp(a)] represent a risk factor for the development of atherosclerotic vascular disease, but the mechanism by which this lipoprotein localizes to involved vessels is only partially understood. In view of studies suggesting a link between inflammation and atherosclerosis and our previous finding that leukocyte defensin modulates the interaction of plasminogen and tissue-type plasminogen activator with cultured human endothelial cells, we examined the effect of this peptide on the binding of Lp(a) to cultured vascular endothelium and vascular smooth muscle cells. Defensin increased the binding of Lp(a) to endothelial cells approximately fourfold and to smooth muscle cells approximately sixfold. Defensin caused a comparable increase in the amount of Lp(a) internalized by each cell type, but Lp(a) internalized as a consequence of defensin being present was not degraded, resulting in a marked increase in the total amount of cell-associated lipoprotein. Abundant defensin was found in endothelium and in intimal smooth muscle cells of atherosclerotic human cerebral arteries, regions also invested with Lp(a). These studies suggest that defensin released from activated or senescent neutrophils may contribute to the localization and persistence of Lp(a) in human vessels and thereby predispose to the development of atherosclerosis.     

Identification and stabilization of large molecular weight PDE-IVs from U937 cells

Cytosolic cyclic nucleotide phosphodiesterases (PDEs) from human (promonocytic) U937 cells were rapidly resolved by DEAE-Sepharose CL-6B anion exchange chromatography into two major peaks of cAMP-specific activity possessing average Kms of 1.70 microM (Peak 1) and 1.65 microM (Peak 2). Both peaks were predominantly PDE-IV, but possessed molecular weights higher than those generally reported for partially purified PDE-IVs. Storage of Peak 2 for 24 h at 4 degrees C resulted in a doubling of its Vmax and an apparent decrease in its molecular weight. Activation of Peak 2 PDE-IV was prevented when the sodium acetate concentration in its buffer was reduced by dilution immediately following isolation. Although the relevance of this activation to cellular regulation of PDE-IV is undefined, the isolation and stabilization of PDE-IV in its large molecular weight form will be critical to future investigations of PDE-IV regulation.     

Demonstration of covalent binding of lipoprotein(a) [Lp(a)] to fibrin and endothelial cells

It has been well documented that Lp(a) binds noncovalently to fibrin or human umbilical vein endothelial cells. This binding is to lysines and is inhibited by lysine analogues such as epsilon-aminocaproic acid (EACA). In the present study, Lp(a) (0.006-0.6 microM) binding to immobilized fibrin and endothelial cells was evaluated by ELISA with an anti-Lp(a) antibody. A significant portion (approximately 65%) of the Lp(a) was found to resist dissociation by EACA (0.2 M). The EACA resistant binding of Lp(a) was time and concentration dependent. The addition of EDTA to the incubation mixture had no effect, thereby excluding cross-linking by transglutaminase as a mechanism. This portion of Lp(a) was also resistant to dissociation by acid (0.1 N HCl), 0.1% SDS, 1 M benzamidine, Tris-HCl (1 M, pH 12), or DTT (5 mM), but it was washed off by 0.1 N NaOH (which did not remove the immobilized fibrin). This suggested that the Lp(a) was covalently linked by an ester bond. Covalent binding was inhibited when Lp(a) was mildly oxidized by BioRad Enzymobeads, which may explain why it escaped recognition in experiments with radiolabeled Lp(a). Covalent binding was attenuated when Lp(a) was pretreated with DFP suggesting that the serine residue in the pseudo active site of Lp(a) was involved. Lp(a) also bound covalently to immobilized BSA, indicating some nonspecificity. However, binding to BSA was almost 3-fold less than to fibrin, suggesting that lysine binding may facilitate covalent binding. A similar proportion of EACA resistant binding of Lp(a) was found with endothelial cells. In conclusion, the findings demonstrate a novel, covalent binding by Lp(a) which is kringle independent and is postulated to involve the pseudo protease domain of Lp(a). This property may contribute to the deposition of Lp(a) on endothelial surfaces and its colocalization with fibrin in atheromas.     

Phospholipid regulation of a cyclic AMP-specific phosphodiesterase (PDE4) from U937 cells

The regulation of phosphodiesterase-4 (PDE4) by various phospholipids was explored using PDE4s partially purified from U937 cells. Preincubation (5 min, 4 degrees C) of the large molecular weight PDE4 denoted "Peak 2 PDE4" with mixed phosphatidic acids (PAs) produced a 2-fold increase in its Vmax without changing its Km (approximately 2 microM) for cyclic AMP. This "activation" was not limited to PAs with specific fatty acid substituents: Synthetic PAs containing saturated and/or unsaturated fatty acids 16-20 carbons long produced similar effects. Lysophosphatidic acids (LPAs) and phosphatidylserines (PSs) also induced PDE4 activation, whereas phosphatidylcholines (PCs), phosphatidylethanolamines (PEs) and diacylglycerol did not. Antibodies to a peptide region near the PDE4 catalytic site specifically inhibited PA-induced activation. The data demonstrate that anionic phospholipids can act as non-essential activators of a leukocyte PDE4, and suggest biochemical crosstalk between phospholipid-dependent and cyclic AMP-dependent signalling pathways in human leukocytes.     

c-Myc plays a role in cellular susceptibility to death receptor-mediated and chemotherapy-induced apoptosis in human monocytic leukemia U937 cells

Human monocytic leukemia U937 cells readily undergo apoptosis when they are treated with TNF-alpha, anti-Fas antibody and anticancer drugs such as etoposide and Ara-C. To study the mechanism of apoptosis, we developed a novel apoptosis-resistant variant, UC, from U937 cells. The UC cells showed resistance to apoptosis induced by TNF-alpha, anti-Fas antibody, etoposide and Ara-C. Somatic cell hybridization between U937 and UC showed that apoptosis-resistance to TNF-alpha in UC was genetically recessive and resistance to etoposide was dominant, suggesting that UC has at least two different mutations functionally involved in apoptosis. Mechanistic analysis revealed that UC cells expressed reduced amounts of c-Myc. Transfection of the c-myc gene into UC cells restored the sensitivity of the cells to undergo apoptosis induced by TNF-alpha and anti-Fas, which attributes apoptosis-resistance in this circumstance to the reduced expression of c-Myc. On the other hand, c-myc transfection into UC cells could not restore their sensitivity to etoposide- and Ara-C-induced apoptosis, arguing against the role of c-myc in chemotherapy-induced apoptosis. However, treating the parental U937 cells with antisense oligonucleotides designed to reduce c-Myc expression rendered the cells resistant to etoposide-induced as well as to TNF-alpha-induced apoptosis. These results indicate that the reduced expression of c-Myc in UC is strongly associated with the resistance to etoposide-induced apoptosis. Our finding that c-myc transfection into UC could not restore the sensitivity to etoposide-induced apoptosis, suggests UC could have a second mutation that confers resistance to etoposide-induced apoptosis in a genetically dominant manner. Taken together, our present results indicate that c-Myc plays a role in cellular susceptibility to death receptor-mediated and chemotherapy-induced apoptosis.     

Proteasome (prosome) subunit variations during the differentiation of myeloid U937 cells

20S proteasomes (prosomes/multicatalytic proteinase) are protein particles built of 28 subunits in variable composition. We studied the changes in proteasome subunit composition during the differentiation of U937 cells induced by phorbol-myristate-acetate or retinoic acid plus 1,25-dihydroxy-cholecalciferol by western blot, flow cytometry and immuno-fluoresence. p25K (C3), p27K (IOTA) and p30/33K (C2) subunits were detected in both the nucleus and cytoplasm of undifferentiated cells. Flow cytometry demonstrated a biphasic decrease in proteasome subunits detection during differentiation induced by RA + VD. PMA caused an early transient decrease in these subunits followed by a return to their control level, except for p30/33K, which remained low. Immuno-fluorescence also showed differences in the cytolocalization of the subunits, with a particular decrease in antigen labeling in the nucleus of RA + VD-induced cells, and a scattering in the cytoplasm and a reorganization in the nucleus of PMA-induced cells. Small amounts of proteasomal proteins were seen on the outer membrane of non-induced cells; these membrane proteins disappeared when treated with RA + VD, whereas some increased on PMA-induced cells. The differential changes in the distribution and type of proteasomes in RA + VD and PMA-induced cells indicate that, possibly, 20S proteasomes may play a role in relation to the mechanisms of differentiation and the inducer used.     

Induction of apoptosis in leukemia U937 cells by 5'-deoxy-5'-methylthioadenosine, a potent inhibitor of protein carboxylmethyltransferase

We found dramatic changes in leukemia U937 cells treated with 5'-deoxy-5'-methylthioadenosine (MTA), a potent inhibitor of protein carboxylmethyltransferase (protein methylase II). Initiation of cell death was observed by 1 day after MTA treatment, and it was induced in a dose- and time-dependent manner. However, cell viability measured by trypan blue exclusion was not consistent with the actual percentage of cell death. These results indirectly indicated that the type of cell death is apoptosis rather than necrosis. Nuclear fragmentation and DNA condensation of MTA-treated U937 cells were analyzed by both fluorescent and electron microscopy. MTA-treated cells first began to arrest in the M phase of the cell cycle, and they then exhibited a mitotic-like nuclear fragmentation process with partially membraneless chromatin. Furthermore, agarose gel electrophoresis of DNA extracted from cells treated with MTA showed DNA laddering with production of fragments of approximately 200 bp multiples. These studies indicated that cell death induced by MTA has the characteristics of apoptosis, although nuclear fragmentation is atypical. It seems likely that the process of apoptosis in U937 cells induced by MTA correlates with incomplete assembly of the nuclear envelope, since MTA itself could inhibit the carboxylmethylation of nuclear lamin B and delayed incorporation of lamin B into the nuclear envelope.     

Thrombin stimulates expression of tissue-type plasminogen activator and plasminogen activator inhibitor type 1 in cultured human vascular smooth muscle cells

The effect of thrombin on the fibrinolytic potential of human vascular smooth muscle cells (SMC) in culture was studied. SMC of different origin responded to thrombin treatment with a dose and time dependent increase in tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor type-1 (PAI-1) levels in both cell lysates and conditioned media with maximum effects achieved at 10-20 IU/ml thrombin. PAI-1 antigen levels also increased in the extracellular matrix of thrombin treated SMC. PAI-2 levels in cell lysates of such SMC were not affected by thrombin. The effect was restricted to active thrombin, since DFP-thrombin and thrombin treated with hirudin showed no increasing effect on t-PA and PAI-1 levels in SMC. Enzymatically active thrombin also caused a four-fold increase in specific PAI-1 mRNA and a three-fold increase in t-PA mRNA. Furthermore we demonstrated the presence of high and low affinity binding sites for thrombin on the surface of SMC with a KD = 4.3 x 10(-10)M and 9.0 x 10(4) sites per cell and a KD = 0.6 x 10(-8) M and 5.8 x 10(5) sites per cell respectively. Thrombin could come in contact with SMC in case of vascular injury or following gap formation between endothelial cells. Our data support the idea that besides its known proliferative effect for SMC, thrombin could also modulate their fibrinolytic system.     

Effects of the synthetic anti-oxidant, probucol, on the U937 monoblastoid cell line

We examined physiological events in the hyperplastic artery, using a method based on the mechanical responsiveness and myosin light-chain phosphorylation in response to various stimulants. Six weeks after endothelial denudation by ballooning of the right carotid artery, strips of this artery with moderate intimal hyperplasia (intimal area was 30-50% of medial area in 20 of 28 rabbits) were used for experiments. Strips from the left carotid served as the normal control. When the hyperplastic artery was stimulated with 30 microM PGF2 alpha, the maximal tension (232.4 +/- 49.1 mg/mg dry wt, mean +/- SD) was significantly higher (P < 0.05) than that of the control (129.5 +/- 16.4 mg/mg). The maximal extent of myosin light-chain monophosphorylation (45.4 +/- 8.9%) and diphosphorylation (10.9 +/- 5.2%) in the hyperplastic artery was significantly higher (P < 0.05) than that in the control artery (33.0 +/- 4.8 and 4.0 +/- 4.8%, respectively). The monophosphorylation of the myosin light chain in the hyperplastic artery was sustained for up to 20 min, while that in the control artery decreased to the basal level within 20 min. Similar observations were obtained by stimulation with 60 mM K+ or 30 microM norepinephrine. Dose-response curves of the development of tension in the hyperplastic artery to various agonists (K+, PGF2 alpha, norepinephrine) shifted upward the curves for the control artery. These results suggest that qualitative changes in the characteristics of smooth muscle cells may occur in the intimal hyperplastic portion, including a hyperreactive contraction associated with enhanced and sustained phosphorylation of the myosin light chain.     

Characterization of a novel iodocyanopindolol and SM-11044 binding protein, which may mediate relaxation of depolarized rat colon tonus

Studies under blockade of alpha-, beta1-, and beta2-adrenoreceptors revealed a good correlation between the responses of rat colon relaxation of depolarized tonus and of rat adipocyte lipolysis elicited by catecholamines or BRL-37344, a selective beta3-adrenoreceptor agonist, suggesting beta3-adrenoreceptor stimulation. In contrast, SM-11044, a nonselective beta-adrenoreceptor agonist, stimulated colon relaxation more efficiently than lipolysis; its effects were differently antagonized by cyanopindolol with pA2 values of 8.31 in colon and of 7.32 in adipocytes. Binding studies in rat colon smooth muscle membranes using [125I]iodocyanopindolol under blockade of adrenaline and serotonin receptors revealed the existence of a single class of sites (Kd = 11.0 nM, Bmax = 716.7 fmol/mg protein). The specific binding was saturable and reversible and was displaced by SM-11044 but not by BRL-37344, isoproterenol, noradrenaline, adrenaline, serotonin, nor dopamine. This binding site was photoaffinity labeled using [125I]iodocyanopindolol-diazirine. The labeling was prevented by SM-11044 but not by BRL-37344. The amino-terminal amino acid sequences of the high performance liquid chromatography-purified peptides generated by enzymatic and chemical cleavages of the affinity labeled 34-kDa protein confirmed that the novel iodocyanopindolol or SM-11044 binding protein of rat colon smooth muscle membranes is different from known adrenaline, serotonin, or dopamine receptors. Its functional role might include the relaxation of depolarized colon.     

Differential regulations of phosphatidylcholine biosynthesis in U937 cells by inhibitors of protein and tyrosine kinases

OBJECTIVES: Multicentricity of hepatocellular carcinoma (HCC) is attracting a great deal of attention at present. However, few studies have focused on the prognostic comparison between unicentric and multicentric multinodular HCCs. The aim of this study is the reevaluation of histologic criteria of multicentric HCC and a prognostic comparison between the two groups mentioned above. METHODS: Forty-nine cases with intrahepatic multiple nodules of HCC, by gross examination, among 184 consecutive resected HCCs were examined clinicopathologically. These cases were divided into three groups: group A, cases suggestive of multicentric genesis; group B, unicentric cases; and group C, indeterminate cases. Histopathological characteristics and the cumulative survival rates were compared among these groups. RESULTS: Five cases were categorized as group A, 36 cases as group B, and eight cases as group C. Nodules in group A were smaller than 2 cm in diameter, situated discretely and well differentiated, and with neither vascular nor capsular invasion. Most of the nodules lacked a tumor capsule and had an irregular border. In the 36 cases of group B, all main tumors had vascular and/or capsular invasion. The cellularity index was almost the same in all groups. The cumulative survival rate of group A was better than that of group B or group C. CONCLUSIONS: Small multiple nodules of well-differentiated hepatocellular carcinoma without vascular and capsular invasion might be multicentric, and these early detections and operations could result in a fairly good prognosis, despite the multiple HCC nodules.     

Isolation, cloning, and expression of a 70-kilodalton plasminogen binding protein of Borrelia burgdorferi

Surface receptors for plasminogen are expressed by many gram-positive and gram-negative bacteria and may play a role in the dissemination of organisms by binding plasminogen, which upon conversion to plasmin can digest extracellular matrix proteins. Two plasminogen binding proteins have been identified for Borrelia burgdorferi, outer surface protein A and a 70-kDa protein (BPBP). We purified BPBP by plasminogen affinity chromatography and obtained its amino acid sequence by Edman degradation of a tryptic digest. The gene coding for BPBP was isolated from a lambda-ZAP II genomic library with probes developed from sequenced portions of the protein. This gene was expressed in Escherichia coli; the recombinant product was seen by antibody raised against native BPBP and also bound 125I-labeled plasminogen. The experimentally derived amino acid sequences corresponded to the predicted sequence encoded by the BPBP gene. The deduced amino acid sequence for BPBP revealed significant similarity to p30, a 30-kDa protein of B. burgdorferi (54% identity and 65% similarity), to a 60-kDa protein in Borrelia coriaceae (66% identity and 80% similarity), to oligopeptide binding protein A of E. coli (34% identity and 57% similarity), and, more generally, to the periplasmic oligopeptide binding family of proteins.     

In vitro toxicity of surfactants in U937 cells: cell membrane integrity and mitochondrial function

Surface-active agents are components of many drugs and cosmetics. In order to examine their cytotoxic and membrane-toxic potential, various surfactants were examined in U937 cells using the tetrazolium reduction assay EZ4U, a modified XTT test, and the arachidonic acid release test (AART). EZ4U measures the ability of living cells to reduce a colorless tetrazolium salt to an orange water-soluble formazan derivative by mitochondrial dehydrogenases. [3H]arachidonic acid ([3H]AA) is rapidly incorporated into cell membrane phospholipids. Due to membrane disintegration or enzymatic catalysis, it is released into the cell culture medium and can be measured by scintillation technique. The results after 24-hour-exposure are as follow (CC50 microg/ml; RC50 microg/ml): benzalkonium chloride (0.6), Cremophor A25 (1.4; 5.9), sodium cetearyl sulfate (1.6; 19.9), Brij78 (1.6; 7.7), TEGO betaine E (3.0; 19.3), TEGO betaine CKD (4.1; 20.2), TEGO betaine L7 (7.5; 20.0), sodium dodecyl sulfate (7.5; 39.0), Triton X-100 (8.9; 110), polysorbate 80 (48.1; 491), soybean lecithin (7920; 19940).     

Tissue plasminogen activator, plasminogen activator inhibitor-1, and fibrin as indexes of clinical course in cardiac allograft recipients. An immunocytochemical study

BACKGROUND: Tissue-type plasminogen activator (TPA) is the principal activator of plasminogen. Since hemostasis in the microcirculation of allografts is a well-recognized complication of transplantation, we asked (1) whether the distribution and amount of cellular TPA in biopsies of transplanted human hearts are associated with fibrin deposits in and around the microcirculation, (2) whether such changes involve the physiological inhibitors of TPA and plasmin, and (3) whether the presence of these activators and inhibitors of fibrinolysis in tissue is correlated with clinical outcome. METHODS AND RESULTS: We immunocytochemically quantified the presence of fibrin, plasmin, TPA, and the TPA inhibitor PAI-1 in 938 biopsies from 68 consecutive cardiac allografts over a 54-month period. The localization, distribution, and quantification of TPA in arteriolar smooth muscle cells revealed that 35 of the 68 allografts maintained vascular TPA reactivity consistent with time-zero biopsies of autologous donor hearts: this was designated as the normal TPA group. In contrast, 33 of the 68 allografts significantly lost vascular TPA reactivity compared with time-zero biopsies of autologous donor hearts: this was designated as the depleted TPA group. Analysis of sequential biopsies from both groups during 54 months revealed that the mean cumulative quantitative TPA value for the normal TPA group was 1.0 +/- 0.01, whereas the depleted TPA group value was 1.9 +/- 0.02 (P = .0001), and the mean cumulative quantitative fibrin value for the normal TPA group was 1.0 +/- 0.01, whereas the depleted TPA group value was 1.5 +/- 0.05 (P = .0001). Biopsies of allografts in the depleted TPA group contained endothelial reactivity for TPA-PAI-1 complexes, whereas biopsies from the normal TPA group did not. Plasmin-associated molecules were rarely identified in biopsies of the normal TPA group but were present in the depleted TPA group, and the fibrin-to-plasmin ratio in the normal TPA group always was less than the fibrin-to-plasmin ratio in biopsies from the depleted TPA group. Analysis of demographic and risk factors revealed no significant differences between patients in the normal and depleted TPA groups, but none of the 35 patients in the normal TPA group died or were retransplanted, and 13 of the 33 patients in the depleted TPA group died or required retransplantation (P = .0001). CONCLUSIONS: Time-zero hearts (n = 68) and 34 of 38 stable allografts contained immunocytochemically detectable TPA only in vascular smooth muscle cells. Twenty-nine of 30 patients with normal TPA in their time-zero biopsies who subsequently developed a poor clinical outcome were found to have depleted TPA in biopsies evaluated during their first postoperative month and remained depleted throughout the study. Of 33 patients with depleted TPA, 39% died or required retransplantation. Depleted arteriolar TPA associated significantly with vascular and interstitial deposits of fibrin, plasmin, and endothelial TPA-PAI-1 complexes. These findings indicate that hemostatic and fibrinolytic pathways are activated in falling allografts, and they reveal evidence of depleted TPA before clinical or histopathological signs of failure. Patients with such allografts were found to be at high risk of death independently of other widely used clinical/laboratory parameters of prediction.