What is the best biomarker for diagnosis of rejection in heart transplantation?

INTRODUCTION: Acute cellular rejection is a major cause of graft loss in heart transplantation (HT). Endomyocardial biopsy remains the gold standard for its diagnosis, but it is an invasive procedure not without risk. A proinflammatory state exists in rejection that could be assessed by determining plasma levels of inflammatory biomarkers. OBJECTIVE: To analyze the utility of various inflammatory markers, which is most important and what values best classify patients to diagnose rejection. MATERIALS AND METHODS: A prospective study in 123 consecutive cardiac transplant recipients was conducted from January 2002 to December 2006. Fibrinogen protein (Fgp) and function (Fgf), C-reactive protein (CRP), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and sialic acid (SA) determinations were performed at one, two, four, six, nine, and 12 months post-HT at the same time as biopsies. Coronary arteriography and intravascular ultrasound were performed on the first and last follow-up visits. Heart-lung transplants, retransplants, pediatric transplants, patients who died in the first month, and patients who refused consent were excluded. Also excluded were determinations that coincided with renal dysfunction, active infection, hemodynamic instability, or a non-evaluable biopsy. The final analysis included 79 patients and 294 determinations. The correlation between the levels of these biomarkers and the presence of rejection in the biopsy (> or = ISHLT grade 3) was studied. RESULTS: We did not find significant differences in the values of any of the markers analyzed on the six follow-up visits. Only CRP showed significant and sustained differences between the two groups (with and without rejection) from the second follow-up visit (month 2). The area under the curve showed significant differences in Fgp (0.614, p = 0.013), Fgf (0.585, p = 0.05), TNF-alpha (0.605, p = 0.02), SA (0.637, p = 0.002) and mainly CRP (0.765, p = 0.0001). CRP levels below 0.87 mg/dL ruled out rejection with a specificity of 90%. CONCLUSIONS: Among the inflammatory markers analyzed, CRP was the most useful parameter for non-invasive screening of acute cellular rejection in the first year post-HT.

Erythrocyte membrane composition in patients with primary hypercholesterolemia.

There are conflicting results regarding the erythrocyte membrane cholesterol and phospholipid content in patients with primary hypercholesterolemia (PHC), due to methodological problems in obtaining haemoglobin-free ghosts. At the same time, the different units used and the fact that the cholesterol and phospholipids are not expressed in relation with integral protein membrane content, produces contradictory results. We have analysed in 33 patients with PHC (12 male, 31 female) aged 43+/-12 years and in 33 healthy normolipaemic volunteers (9 male, 24 female) aged 43+/-13 years plasma lipids, along with, erythrocyte membrane cholesterol, phospholipids and integral proteins. PHC patients showed increased erythrocyte membrane cholesterol: 0.36+/-0.15 mg/mg when compared with controls: 0.29+/-0.75 mg/mg; p=0.018. Phospholipid membrane content, although higher in the cases, did not reach statistical significance (PHC patients: 0.38+/-0.15 mg/mg vs. 0.33+/-0.72 mg/mg; p=0.098). The cholesterol/phospholipids ratio (Chol/Ph) was 0.99+/-0.22 in PHC patients versus 0.92+/-0.28 in controls; p=0.127. Our results suggest that there is a slight increase in erythrocyte membrane cholesterol in patients with PHC. Given the increasing importance of erythrocyte membrane cholesterol in the stability of the atheroma plaque due its possible contribution to the clinical signs of ischaemic heart disease, it seems relevant to determine this parameter in risk populations. Therefore, a simple and reproducible method needs to be standardised which would enable comparisons between laboratories and facilitate further studies aimed to it as a marker of acute coronary syndromes.

Erythrocyte deformability in obesity measured by ektacytometric techniques.

Erythrocyte deformability (ED) has been scarcely evaluated in obese patients without other concomitant cardiovascular risk factors and contradictory results have been published regarding the influence of plasma lipids on the erythrocyte membrane lipid composition and insulin resistance on this rheological parameter. In 67 severe or morbid obese patients without other cardiovascular risk factors (51 women and 11 men, aged 34+/-11 years) and in 67 controls (45 women and 22 men, aged 32+/-10 years), ED has been determined by ektacytometric techniques in a Rheodyn SSD, the elongation index (EI) being measured at 12, 30 and 60 Pa, along with plasma lipids, red blood cell membrane lipids (cholesterol and phospholipids) and insulin resistance indexes in basal conditions and after a three month diet period. No significant differences were obtained in the EI between obese patients and the control group at any of the shear stresses tested (P>0.05). The cholesterol and phospholipid content of the red blood cell membrane did not significantly differ between cases and controls (P>0.05). Obese patients with metabolic syndrome showed lower EI at 30 and 60 Pa than those without metabolic syndrome (P=0.014 and P=0.031 respectively). Weight loss was not accompanied by any changes in these rheological parameters. Obesity itself does not seem to modify ED. However, metabolic syndrome seems to decrease ED, possibly through insulin resistance.

Usefulness of von Willebrand factor in cardiac allograft vasculopathy: preliminary experience.

BACKGROUND: Cardiac allograft vasculopathy (CAV) is a disease that significantly limits the survival of transplant patients intravascular ultrasound (IVUS) is considered the method of choice for its diagnosis. von Willebrand factor (vWf) has been used as a marker of endothelial malfunction. We sought to evaluate the usefulness of vWf as a CAV marker. MATERIALS AND METHODS: We prospectively analyzed 22 cardiac transplant subjects, on whom we performed a first study using coronary angiography and IVUS at 36 +/- 3 days and a second study at 598 +/- 49 days. During the follow-up period, five vWf serum controls were performed per patient. We analyzed the results with the repeated-measures ANOVA test and a ROC curve. RESULTS: CAV was detected in 10 (45.5%) of the 22 patients. Although vWf levels tended to diminish progressively during evolution, this trend was not statistically significant (P = .3). However, differences were appreciated based on the presence versus absence of CAV (298 +/- 139 mg/dL versus 212 +/- 105 mg/dL, P = .02). The ROC curve showed a sensitivity of 40%, a specificity of 83%, and a negative predictive value of 82% with a cutoff point of 300 mg/dL. CONCLUSIONS: Subjects with CAV showed significantly higher vWf serum concentrations, particularly during the preliminary phases of cardiac transplantation decreasing during its evolution. This marker could be useful for early screening of CAV.

Diagnostic usefulness of inflammatory markers in acute cellular rejection after heart transplantation.

BACKGROUND: Acute cellular rejection (ACR) affects early morbidity and mortality after heart transplantation. The diagnostic technique of choice is endomyocardial biopsy. Our aim was to evaluate the diagnostic usefulness of inflammatory markers as a noninvasive method to monitor cellular rejection. MATERIAL AND METHODS: We prospectively analyzed 73 cardiac transplant patients by determining the serum levels of protein fibrinogen (fgpro), functional fibrinogen (fgfun), C-reactive protein (CRP), and sialic acid (SA) coinciding with an endomyocardial biopsy (5.1 revisions/patient). The statistical methods were chi(2), Student's t-test, and ROC curves. RESULTS: Of the 373 controls, significant rejection was detected in 19%. Analysis of the relationship between ACR and the markers showed significantly elevated levels of fgpro (345 +/- 90 versus 307 +/- 74 mg/dL; P = .03), fgfun (361 +/- 101 versus 318 +/- 89 mg/dL; P = .04), and SA (74 +/- 22 versus 66 +/- 15 mg/dL; P = .02), but not CRP (19 +/- 29 versus 10 +/- 21 mg/dL; P = .07). SA displayed a better diagnostic utility (area under the curve 0.7; P < .01), 35% sensitivity, 85% specificity, and 82% negative predictive value for a cutoff point of 80 mg/dL. CONCLUSIONS: Among the inflammatory markers increased in ACR, SA was the most useful noninvasive tool for screening.

Nonhistone protein changes during the diethylnitrosamine-induced carcinogenesis of rat liver.

Diethylnitrosamine (DENA) was administered with drinking water (40 mg/liter) to male Wistar rats for 4, 6, 8, and 10 weeks. The protein:DNA ratio and the ultraviolet light spectral properties of liver chromatin were not modified by DENA treatment. Nonhistone proteins were separated by sodium dodecyl sulfate:polyacrylamide electrophoresis on slab gels and analyzed by densitometry with a scanning microphotometer connected on line to a computer. There were no qualitative changes in the pattern of nonhistone proteins during the treatment with DENA. The quantitative changes statistically significant at p less than 0.005 were detected only in the 4th and 10th week, increases in fractions with molecular weights of 41,000 to 47,000 and 51,000 to 64,000 and decreases in fractions with molecular weights of 27,000 to 32,000 and 47,000 to 51,000 having been found. The proteinase activity of liver chromatin was assayed in incubation mixtures with 0.2 and 2 M NaCl and the measurement of the cleavage products was performed with ninhydrin. Proteolytic activity was found only in 0.2 M NaCl and was higher in rats treated for 8 weeks with DENA than in controls. Autolysis of chromatin for 24 hr at 37 degrees showed a severe breakdown of the nonhistone protein, being greater in the high-molecular-weight fractions than in the low-molecular-weight fractions.

Elevated high molecular weight fibrinogen in plasma is predictive of coronary ischemic events after acute myocardial infarction.

This study investigates the association between the concentration and function of plasma fibrinogen molecules measured at the time of hospital admission in patients with acute myocardial infarction (AMI), with reference to the risk of new coronary ischemic events during a three-day follow-up period of. Before starting fibrinolytic and anticoagulant treatment plasma fibrinogen, high molecular weight fibrinogen (HMW-fibrinogen), fibrin formation rate (FbFR) and phosphorous content in fibrinogen were determined in 90 AMI patients. During a three-day follow-up period 12 patients suffered new ischemic events. The 12 patients with coronary ischemia had higher concentrations of plasma fibrinogen (312+/-23 vs. 270+/-73 mg/dl, p<0.05) and HMW-fibrinogen (246+/-35 vs. 189+/-23 mg/dl, p<0.001) and a higher FbFR (65+/-30 vs. 40+/-25, p<0.001) than patients without these events. No association was found between the phosphorous content in fibrinogen and new coronary ischemic events. We conclude that after myocardial infarction an elevated plasma level of HMW-fibrinogen and a high FbFR value at the time of hospital admission are associated with new coronary ischemic events during a three-day follow-up period.

Dysfunctional plasminogen in full term newborn--study of active site of plasmin.

The functional activity and active site of plasmin in full-term newborns have been studied and compared to those in adults in order to investigate the nature of the abnormality found in newborn plasminogen described in a previous paper. The functional activity of newborn plasminogen measured on chromogenic substrate was approximately 18% that of adult plasminogen when streptokinase was used as an activator and 12% when urokinase was used. Proteolysis of newborn plasminogen by urokinase yielding a two-chain plasmin form occurred normally, but the incorporation of diisopropylphosphorofluoridate into the light chain of newborn plasmin was approximately 23% of that observed in the light chain of adult plasmin. These observations suggest that the abnormality of full-term newborn plasminogen is located in the active site of the molecule.

Arachidonate incorporation into rat aorta lipid fractions and eicosanoid formation. Effect of prolonged prostacyclin production.

Endogenous arachidonic acid (AA) content, incorporation of radiolabelled AA (AA*) into total lipids, main lipid fractions and different phospholipids (PL), and prostanoid formation have been evaluated in fresh (control) rat arteries and in arteries after 180 min of incubation in buffer (exhausted). The results show that PGI2 formation from endogenous AA decreased 90% in exhausted arteries while AA content decreased only 30%. The total AA* incorporation was significantly higher in exhausted arteries than in controls (p less than 0.01). The distribution of AA* in lipids is altered in exhausted arteries; it increases in total PL, particularly in phosphatidylethanolamine, and decreases in phosphatidylcholine and phosphatidylserine + phosphatidylinositol. AA* content was also lower in triglycerides and esterified cholesterol of exhausted arteries than in control arteries. The AA* metabolized to PGI2 was 83% lower in exhausted arteries than in controls, while PGE2 and TXB2 formation were not modified by the exhaustion process. When the effect that longer incubation in plasma (180 min) has on AA metabolism and turnover was evaluated, PGI2 formation from endogenous AA was found to be increased in comparison with arteries incubated for the de same period in buffer, and the changes observed in the distribution of AA in lipid fractions are smaller than those found in buffer-exhausted aortas. The results of the present study indicate that prolonged production of prostanoids leads to an alteration in AA turnover and to an inactivation of the PGI2-forming system. Plasma seems to protect AA metabolism.

Relationship between fibrinogen protein and fibrinogen function in postmyocardial infarction patients.

The present study investigates the association between increases in the concentration and function of plasma fibrinogen in two groups of patients with chronic ischemic heart disease (11 with recurrent ischemic events and 19 free of these episodes) and in 34 healthy controls. The fibrinogen function index (fibrinogen function per unit of fibrinogen protein) (FgFI) was used as a measure of the fibrinogen clotting potential. The prothrombin fragment 1+2 (F1+2) and thrombin-antithrombin (TAT) were used as procoagulant markers. Plasma sialic acid (SA) was also evaluated as an inflammatory marker. No differences were found between FgFI (1.06+/-0.13 vs. 1.02+/-0.13), F1+2 (1.2+/-0.5 vs. 1.1+/-0.4 nmol/l) and TAT (2.5+/-1.3 vs. 2.5+/-0.7 microg/ml) in postinfarction patients without recurrent coronary ischemic events and the control group. However, postinfarction patients who suffered recurrent coronary ischemic events had significantly higher FgFI than patients without these symptoms (1.19+/-0.09 vs. 1.06+/-0.13), P<.01) and than the control group (1.19+/-0.09 vs. 1.02+/-0.13, P<.001). Moreover, the F1+2 (1.4+/-0.5 vs. 1.1+/-0.4 nmol/l, P<.05) and TAT (3.6+/-3.3 vs. 2.5+/-0.7 microg/ml, P<.05) were significantly higher in patients who suffered recurrent coronary ischemic events than in the control group. However, F1+2 and TAT were not different between patients with and without these symptoms. The fibrinogen protein (Fg-protein) concentration and high molecular weight fibrinogen (HMW-Fg) levels were significantly higher in both postinfarction patient groups than in the control group and in postinfarction patients with recurrent coronary ischemic events than in postinfarction patients without these symptoms. The plasma SA levels were significantly increased in postinfarction patients with and without recurrent coronary ischemia as compared with the control group. A positive correlation was found between fibrinogen and SA levels (r=.5, P<.01). In conclusion, our study indicates that the procoagulant factors, among which we include fibrinogen, F1+2 and TAT play a very active role in recurrent ischemic events in postmyocardial infarction patients. High plasma concentrations of both fibrinogen and SA suggests that fibrinogen becomes elevated as a consequence of inflammatory processes. The FgFI as an indicator of clotting potential of fibrinogen appears to be associated with ischemic events in chronic coronary artery disease.

VEGF and TSP1 levels correlate with prognosis in advanced non-small cell lung cancer.

PURPOSE: There is a need for biomarkers that may help in selecting the most effective anticancer treatments for each patient. We have investigated the prognostic value of a set of angiogenesis, inflammation and coagulation markers in patients treated for advanced non-small cell lung cancer. PATIENTS AND METHODS: Peripheral blood samples were obtained from 60 patients before first line platinum-based chemotherapy ± bevacizumab, and after the third cycle of treatment. Blood samples from 60 healthy volunteers were also obtained as controls. Angiogenesis, inflammation and coagulation markers vascular endothelial growth factor (VEGF), their soluble receptors 1 (VEGFR1) and 2 (VEGFR2), thrombospondin-1 (TSP-1), interleukin-6 (IL6), sialic acid (SA) and tissue factor (TF) were quantified by ELISA. RESULTS: Except for TSP-1, pre- and post-treatment levels of all markers were higher in patients than in controls (p < 0.05). There was a positive and significant correlation between VEGF and VEGFR2 before treatment. VEGF also correlated with inflammatory markers IL-6 and SA. Moreover, there was a positive and significant correlation between levels of VEGFR1 and TF. Decreased levels of TSP-1 and increased levels of VEGF were associated with shorter survival. Bevacizumab significantly modified angiogenesis parameters and caused a decrease of VEGF and an increase of TSP-1. CONCLUSION: Angiogenesis, inflammation and coagulation markers were increased in NSCLC patients. Increased levels of VEGF and low levels of TSP-1 correlated with a poor prognosis.

Prostacyclin production and lipid peroxidation in the aorta of rats fed with cholesterol autoxidation products.

The stable metabolite of prostacyclin 6-keto PGF1 alpha originating from the (1-14C) arachidonic acid, and lipid peroxidation expressed as thiobarbituric acid-reacting substance (TBARS) were studied in the aorta of rats fed a diet enriched in 2% w/w cholesterol autoxidation products for 24 hours prior to sacrifice. A slight increase was found in the amount of TBARS as well as in the conversion of (1-14C) arachidonic acid to 6-keto PGF1 alpha. These results suggest that in aorta there exists a protection mechanism which acts to increase the production of prostacyclin.

Unstimulated and thrombin-stimulated platelets binding to immobilized fibrinogen and fibrin on polystyrene supports.

The binding of unstimulated and thrombin-stimulated platelets was studied with immobilized fibrinogen and fibrin on polystyrene. The amount of fibrinogen bound to the polystyrene support was 2 micrograms/tube, which represents 2.35 micrograms/cm2. Immobilized fibrin was obtained by adding thrombin (5 nM) to immobilized fibrinogen. The number of unstimulated 111In-platelets bound to immobilized fibrinogen and fibrin was similar (3.2 +/- 0.3 x 10(6) and 3.1 +/- 0.4 x 10(6) platelets/micrograms fibrin(ogen), respectively). The platelet binding steadily increased. In the first 2 min, the binding rate was 0.23 x 10(6) platelets/micrograms fibrinogen/min. The binding rate then increased rapidly and saturation was reached at 10 min. The extent of the adhesion of resting platelets to immobilized fibrinogen is about one half that of the same platelets stimulated with thrombin. In thrombin-stimulated 111In-platelets, the binding to immobilized fibrinogen and fibrin is time dependent, and saturation is reached at 5 min. The early rate of thrombin-stimulated platelet binding to fibrinogen is about twice that of binding to fibrin (1.25 and 0.74 x 10(6) platelets/micrograms fibrin(ogen)/min, respectively). In saturation conditions, 1 microgram fibrinogen binds 5.7 +/- 0.6 x 10(6) thrombin-stimulated platelets and 1 microgram fibrin binds 4.6 +/- 0.5 x 10(6) thrombin-stimulated platelets. Our results indicate that the rate of platelet aggregation is faster than fibrin formation, and the rate of fibrinogen-platelet binding is faster than that of fibrin-platelet binding. Therefore, after thrombin stimulation, the binding of platelets to fibrin must be secondary to the binding of platelets to fibrinogen.

The effect of thrombin on the dynamic exchange between intraplatelet and extraplatelet fibrinogen.

We examined the distribution of platelet fibrinogen and the exchange between intra- and extra-platelet fibrinogen in unstimulated and thrombin-stimulated platelets. In unstimulated platelets 60% of platelet fibrinogen was found in the soluble platelet fraction and 40% in the insoluble one. In platelets activated with thrombin, changes took place in the distribution of intraplatelet fibrinogen but not in the total fibrinogen content. At > or = 0.5 U/ml of thrombin the fibrin(ogen) content of the insoluble and soluble fractions was approximately 80% and 20%, respectively. When we evaluated how extraplatelet fibrinogen affects the content and distribution of intraplatelet fibrinogen, we found that when unlabelled fibrinogen was added to unstimulated and thrombin-stimulated platelets the content and distribution of intraplatelet fibrinogen remained unaltered. However, when 125I-fibrinogen was added, it was incorporated into unstimulated and thrombin-stimulated platelets. In unstimulated platelets, 70% of the incorporated 125I-fibrinogen was in the soluble fraction and 30% in the insoluble. In thrombin-stimulated platelets the distribution of the incorporated 125I-fibrinogen was 62% and 38% in soluble and insoluble fractions respectively. MoAb to GPIIb-IIIa produced 80% and 60% inhibition of 125I-fibrinogen incorporation by unstimulated and thrombin-stimulated platelets. Our data showed dynamic exchange between intraplatelet and extraplatelet fibrinogen both in unstimulated and thrombin-stimulated platelets mediated mainly by GPIIb-IIIa.

Lack of effect of thrombin on fibrin(ogen)-endothelial cell interaction.

In the present study we investigate the fibrin(ogen)-endothelial cell binding and the effect of thrombin on the endothelial cells in relation to fibrin(ogen) binding capacity. Endothelial cell fibrinogen binding was concentration and time-dependent, reaching saturation at 1.4 µM of added ligand. At equilibrium, the number of fibrinogen molecules bound per endothelial cell in the monolayer was 5.8±0.7×10(6). When endothelial cells were activated by different concentrations of thrombin (0-0.1 NIH units ml(-1)), no increase in fibrinogen binding capacity was observed at all the thrombin concentration tested. Whereas disruption of endothelial cell monolayers was observed at thrombin concentrations higher than 0.05 NIH units ml(-1), no increase in the amount of fibrinogen bound was observed. Therefore, resting and thrombin-activated endothelial cells show the same fibrinogen binding capacity.The adhesion of endothelial cells in suspension on immobilized fibrinogen or fibrin was studied to ascertain whether the behavior of fibrin is similar to that of fibrinogen. The extent of endothelial cell attachment to immobilized fibrinogen and fibrin was similar (4275±130 cells cm(-2) for fibrinogen and 4350±235 cells cm(-2) for fibrin) and represent approximately 40% of the added endothelial cells. However, endothelial cell adhesion to immobilized fibrin was significantly faster than endothelial cell adhesion to immobilized fibrinogen. The maximum binding rate was 66±9 and 46±8 cells cm(-2) min(-1) for fibrin and fibrinogen, respectively. Therefore, the fibrinopeptides released by thrombin from fibrinogen induce qualitative changes which enhance the fibrin interaction with the endothelial cells.

Characteristics of arachidonic acid metabolism of human endothelial cells in culture.

Human umbilical endothelial cells in culture retain differentiated morphological and functional characterization in primary culture and even in the early subcultures, after which they begin to degenerate. We have studied the morphological and biochemical characterization (ability to produce prostacyclin, prostaglandin E2 and thromboxane A2 in culture) of endothelial cells in the first seven subcultures. In addition the influence of serum and endothelial cell growth factor added to the culture medium have been evaluated. With 20% normal human serum, cell proliferation is faster than with the same concentration of human fetal or bovine fetal serum. After the 3rd passage, morphological and growth alterations become observable in the endothelial cells. However, prostacyclin, prostaglandin E2 and thromboxane A2 production showed no variations during the study.