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Blood, Vol. 93 No. 9 (May 1), 1999:
pp. 2945-2950
Vitamin E Reduces Monocyte Tissue Factor Expression in Cirrhotic
Patients
By
Domenico Ferro,
Stefania Basili,
Domenico Praticó,
Luigi Iuliano,
Garret A. FitzGerald, and
Francesco Violi
From the Department of Internal Medicine, I Clinica Medica, and
Department of Therapeutic Medicine, University "La Sapienza,"
Rome, Italy; and the Center for Experimental Therapeutics, University
of Pennsylvania, Philadelphia, PA.
 |
ABSTRACT |
Clotting activation may occur in liver cirrhosis, but the
pathophysiological mechanism has not been fully elucidated. Because a
previous study demonstrated that lipid peroxidation is increased in
cirrhosis, we analyzed whether there is a relationship between lipid
peroxidation and clotting activation. Thirty cirrhotic patients (19 men
and 11 women; age, 34 to 79 years) and 30 controls matched for sex and
age were investigated. In all subjects, monocyte expression of tissue
factor (TF) antigen and activity; plasma levels of prothrombin fragment
1+2 (F1+2), a marker of thrombin generation; and urinary excretion
of Isoprostane-F2 -III, a marker of lipid peroxidation, were measured. Furthermore, the above-reported variables were re-evaluated after 30 days of treatment with standard therapy (n = 5)
or standard therapy plus 300 mg vitamin E twice daily (n
= 9). In addition, we analyzed in vitro if vitamin E (50 µmol/L) influenced monocyte TF expression and F1+2 generation. Cirrhotic patients had higher values of Isoprostane-F2-III
(P < .0001), F1+2 (P < .0001), and monocyte TF
antigen (P < .0001) and activity (P < .03) than
controls. Isoprostane-F2-III was significantly
correlated with F1+2 (Rho = 0.85; P < .0001) and TF
antigen (Rho = 0.95; P < .0001) and activity (Rho = 0.94; P < .0001). After vitamin E treatment,
Isoprostane-F2-III (P = .008), F1+2
(P < .008), and monocyte TF antigen (P = .012) and
activity (P = .008) significantly decreased; no changes of these variables were detected in patients not receiving vitamin E. In
vitro, vitamin E significantly reduced the expression of monocyte TF
antigen ( 52%; P = .001) and activity (55%; P
= .003), as well as F1+2 generation (51%; P = .025).
This study shows that vitamin E reduces both lipid peroxidation and
clotting activation and suggests that lipid peroxidation may be an
important mediator of clotting activation in liver cirrhosis.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
AMONG THE COMPLEX hemostatic disturbance
occurring in liver cirrhosis (LC), hyperfibrinolysis is considered one
of the most important factors that might favor
hemorrhage.1-4 Hence, the elucidation of the mechanism
leading to hyperfibrinolysis would help us to explore new therapeutic
strategies to prevent bleeding. Recently, we provided evidence that in
LC patients hyperfibrinolysis is secondary to an ongoing prothrombotic
state, which was documented by elevated circulating levels of the
prothrombin fragment 1+2 (F1+2), a marker of in vivo thrombin
generation.5 Clotting activation was particularly evident
in patients with moderate-severe liver failure, suggesting a role for
liver dysfunction in accelerating thrombin generation rate.
Cross-sectional as well as interventional study provided evidence that
endotoxemia, which is elevated in cirrhosis as a consequence of
impaired hepatic clearance,6 might play a pivotal role.
Thus, in cirrhotic patients receiving oral nonabsorbable antibiotics,
the decrease of endotoxemia was closely associated with a significant
reduction of F1+2 plasma levels.5
The relationship between clotting activation and endotoxemia has been
recently investigated by measuring the monocyte expression of tissue
factor (TF). We found that cirrhotic patients have an enhanced monocyte
TF expression that correlated significantly with
endotoxemia.7 Even if a cause-effect relationship between endotoxemia and TF expression was not documented in cirrhotic patients,
several clinical and experimental models demonstrated that endotoxemia
enhances the monocyte TF expression.8-10 However, an
important still-open issue was to analyze the mechanism allowing endotoxemia to enhance TF expression and, in turn, clotting activation. Recent studies demonstrated that bacterial lipopolysaccharide (LPS)
enhances monocyte formation of oxidant species,11,12 suggesting that oxygen free radicals, in particular peroxides, may
represent an important intracellular mechanism by which LPS activates
monocytes. Consistently, several antioxidant agents have been proved to
decrease in vitro LPS-mediated monocyte TF expression.13,14
We have recently showed that lipid peroxidation is enhanced in
cirrhosis and that endotoxemia could play an important role.15 Isoprostane-F2 -III was used as a
marker of lipid peroxidation, because it is elevated in clinical
settings associated with in vivo oxidant stress16,17 and is
generated during low density lipoprotein (LDL) oxidation
in vitro in coincidence with lipid peroxides
formation.18,19 Therefore, we performed a cross-sectional study as well as an interventional study, using vitamin E as an antioxidant agent, to investigate whether a relationship between lipid
peroxidation and clotting activation does exist in patients with cirrhosis.
| |
MATERIALS AND METHODS |
Subjects
Thirty consecutive patients with hepatic cirrhosis (11 women and 19 men; age, 60 ± 11 years; age range, 34 to 79; 15 current smokers)
and 30 healthy volunteers (18 men and 12 women; age, 55 ± 9 years;
age range, 40 to 75 years; 10 current smokers) were studied. The
diagnosis of cirrhosis was established by liver needle biopsy in all
patients. All patients showed normal renal function. Patients were
excluded from consideration if they had: (1) hepatocarcinoma, diagnosed
by the combination of hepatic ultrasound and/or computed tomography
together with elevated serum levels of  -fetoprotein; (2) spontaneous
bacterial peritonitis or other infectious diseases, as indicated by
clinical signs (fever and/or abdominal pain) and attendant (ascitic and
blood culture, polymorphonuclear count in ascitic fluid) indexes; or
(3) cholestatic liver disease.
The Internal Medicine Review Board of the University Hospital of Rome
approved the study. All subjects gave informed consent to their
inclusion in the study. All abstained from nonabsorbable antibiotics
and vitamin supplements for 30 days before the study. In case of
immediate need for blood or plasma, patients were excluded from the
study. The degree of liver failure was scored as mild (class A; n = 6),
moderate (class B; n = 20), or severe (class C; n = 4) according to the
Child-Pugh's criteria, based on clinical (ascites, encephalopathy) and
laboratory (albumin, bilirubin, prothrombin time) parameters, as
previously described.5 The etiology of cirrhosis was
post-hepatitis B in 10 (33%) patients, post-hepatitis C in 18 (60%)
patients, and postalcoholic in 2 (7%) patients.
Design of the Studies
In vivo study.
In a first study, a cross-sectional analysis including the measurement
of monocyte TF expression, plasma levels of prothrombin F1+2,
endotoxemia, and urinary excretion of Isoprostane-F2-III was performed in cirrhotic patients and controls.
A second study was designed to explore the effect of vitamin E
supplementation on monocyte TF expression and systemic clotting activation. To this purpose, 14 of 30 cirrhotic patients (8 women and 6 men; age, 42 to 75 years; 1 of A, 9 of B, and 4 of C class) who gave
informed consent to participate also in this study received 300 mg
vitamin E twice daily plus standard treatment (n = 9) or continued standard treatment (n = 5) for 30 days. Standard treatment consisted of spironolactone, furosemide or ethacrinic acid, albumin, and lactulose. The two groups were homogeneous for sex, age, and degree
of liver failure. Monocyte TF expression, F1+2 plasma levels, and
urinary excretion of Isoprostane-F2-III were evaluated before and at the end of the treatment period.
In vitro study.
The study was performed to analyze the effect of vitamin E coincubation
on LPS-stimulated monocyte TF expression.
Monocytes taken from healthy subjects were preincubated with or without
50 µmol/L vitamin E (-tocopherol; Sigma-Aldrich, Milan, Italy) for
1 hour and were then stimulated with 0.4 ng/mL LPS (Escherichia
coli OB11: B4; Sigma, St Louis, MO). TF expression and thrombin
generation were measured over 6 and 24 hours of incubation time,
respectively, as described below.
Methods
Blood coagulation study.
Blood samples anticoagulated with sodium citrate (9:1, vol/vol) were
taken from patients who had fasted for at least 12 hours between 8:00
and 9:00 AM. The samples were immediately centrifuged at
2,000g for 20 minutes at 4°C, and the supernatant was
collected and stored at 80°C until measurement. Plasma
levels of human prothrombin fragment F1+2 were assayed by an enzyme
immunoassay based on the sandwich principle (Enzygnost F1+2;
Behringwerke, Marburg, Germany; reference value, 0.6 ± 0.2 nmol/L; range, 0.3 to 1.2 nmol/L).4 Intra-assay and
interassay coefficients of variation were 8% and 9%, respectively.
Endotoxemia.
The test was performed using a chromogenic substrate test (Kabi
Diagnostica, Stockholm, Sweden) employing the end-point method, as
previously described.5 The day-to-day coefficient of
variation was 11% (reference value, 4.4 ± 1.5 pg/mL).
Measurement of Isoprostane-F2-III.
Urinary Isoprostane-F2-III levels were assayed using a
stable-isotope dilution gas chromatography/mass spectrometry assay as
already described.20 Briefly, a known amount of the
internal standard [18O2]
Isoprostane-F2-III, prepared as previously described by
Pickett and Murphy,21 was added to each sample. After
solid-phase extraction, the samples were purified by thin-layer
chromatography and analyzed on a Fison M800 (Fison Instruments, Milan,
Italy) gas chromatography/mass spectrometer. Quantification was
performed using peak area ratios. Urinary creatinines were determined
by a standard automated colorimetric assay using a Beckman Synchron CX
System (Beckman Instruments, Arlington Heights, IL).
Isolation and incubation of blood mononuclear cells.
Peripheral blood mononuclear cells were isolated from the heparinized
venous blood of liver cirrhotic patients and controls using aseptic
technique. Platelets were removed by using two-step centrifugation,
once at 140g and twice at 100g in phosphate-buffered saline (PBS) at room temperature for 10 minutes. Peripheral blood mononuclear cells (PBMCs) were isolated by centrifugation on lymphoprep (Nyegaard, Oslo, Norway) at 1,200g for 20 minutes at 20°C.
Monocytes, identified by May-Grunwald-Giemsa staining, comprised 16%
to 22% (mean, 19%).
Monocytes (adherent cells) were obtained by incubation of the PBMCs for
90 minutes at 37°C in humidified atmosphere of 5% CO2
in air in Petri dishes containing RPMI 1640, supplemented with 2 mmol/L
glutamine; lymphocytes (nonadherent cells) were removed by aspiration
with a Pasteur pipette and washing of the dishes with warm
media.22 The purified monocyte preparation contained 85%
to 95% monocytes. After isolation, cells were washed twice in PBS and
incubated without LPS at 2 × 105 cells/mL in RPMI
1640 at 37°C 5% CO2 for 6 hours. At the end of the
incubation period, the cells and media were separated by centrifugation
(2,000g for 15 minutes). The cells were washed with Tris-NaCl
buffer (0.1 mmol/L NaCl, 0.1% bovine serum albumin, pH 7.4) and then
lysed in the same buffer by adding 15 mmol/L n-octyl- -D-glycopyranoside at 37°C for 30 minutes.9
A cell count and trypan blue exclusion were performed on cell
suspensions after washing.
In another set of experiments (see in vitro study above), monocytes
(2 × 105 cells/mL) taken from healthy
volunteers were preincubated for 1 hour with 50 µmol/L vitamin E or
medium as control and then incubated with 0.4 ng/mL LPS in RPMI 1640 at
37°C in 5% CO2 for 6 hours. At the end of incubation
period, the samples were treated as described above.
TF assay.
TF activity was determined in the cell lysate by measuring monocyte
procoagulant activity with a one-stage clotting assay.23 Briefly, aliquots (100 µL) of cell lysate were added to 100 µL of
normal pooled citrated plasma; after 150 seconds of incubation at
37°C, 100 µL of 0.025 mmol/L CaCl2 was added and the
clotting time was recorded using a Schnitger and Gross
coagulometer (Germany). All samples were tested in
duplicate. Clotting times were converted to arbitrary TF units per 2 × 105 monocytes using logarithmic plots of clotting
times versus dilution of a standard TF solution obtained using
commercial thromboplastin (Dade International Inc, Miami,
FL). Undiluted thromboplastin was assigned a value of 1,000 TF units,
corresponding to a clotting time of 14 seconds. This procoagulant
activity was not demonstrated with plasma deficient in factors
VII, X, or V.
The enzyme-linked immunosorbent assay (ELISA) for measuring TF antigen
in cell lysate was performed using a commercial kit (Imubind Tissue
factor Elisa Kit; American Diagnostica Inc, Greenwich, CT). The lower
detection limit is approximately 10 pg/mL. The assay recognizes
TF-apolipoprotein (TF-apo), TF, and TF-factor VII (TF-VII)
complexes and is designed such that there is no interference from other
coagulation factors or inhibitors of procoagulant activity.
Thrombin generation rate by LPS-stimulated monocytes.
Thrombin generation rate by LPS-stimulated monocytes was evaluated in
vitro, by incubating LPS-stimulated monocytes with heparinized standard
plasma. For this purpose, a low molecular weight heparin (LMWH)
anticoagulated blood sample (ratio, 1:10) was taken from healthy
volunteers who gave informed consent to participate in the study. The
blood was anticoagulated with LMWH (20 U/mL), because this agent
effectively inhibits the formation of thrombin in solution but has only
a small effect on thrombin generated at and bound to
surfaces.24 Monocytes (2 × 105 cells/mL)
taken from healthy subjects were preincubated for 1 hour with vitamin E
(50 µmol/L) or medium as control and then incubated with LPS (0.4 ng/mL) for 6 hours as described above. The medium was removed and 1,000 µL of overlay heparinized standard plasma was added to each well and
incubated at 37°C for 24 hours.25 After the incubation,
samples were harvested, centrifuged at 4,000g, and assayed for
F1+2 generation, which was calculated from the increase in F1+2 level
compared with value obtained in control samples, which consisted of
heparinized plasma added with LPS. All samples were assayed in
duplicate. The results represent the mean of four experiments.
Statistical Analysis
Statistical analysis was performed using the  2 statistic
or Fisher's Exact Test for independence. Pairwise analysis was
performed as appropriate. Two-tailed tests of significance were used
throughout. Correlations were assessed using linear regression
analysis. When necessary, log transformation was used to normalize the
data, or appropriate nonparametric tests were employed. Data are
presented as the median (with the range in parentheses) given the
apparent departure of data from distributional normality. The required significance level for all tests was set at .05.26
| |
RESULTS |
Urinary excretion of Isoprostane-F2-III
(median [range]: 256 [44-812] v 80 [49-160] pg/mg
creatinine; P < .0001), F1+2 plasma levels (mean [standard
deviation (SD)]: 1.7 [0.7] v 0.6 [0.25] nmol/L; P < .0001), monocyte TF antigen (median [range]: 41.5 [5-101.8]
v 15.5 [10-39] pg/2 × 105 monocytes;
P < .0001), and activity (median [range]: 17.5 [0-50] v 9 [4-20] U/2 × 105 monocytes;
P < .03) were significantly higher in cirrhosis patients than
in controls.
The clinical and laboratory characteristics of liver cirrhotic patients
divided according to Child-Pugh classification are reported in
Table 1. TF antigen (P = .0009) and
activity (P = .0034) and prothrombin fragment F1+2 (P = .0062) progressively increased from A to C class (Table 1). A
significant correlation was observed between prothrombin fragment F1+2
and TF activity (Rho = 0.89, P < .0001) and antigen (Rho = 0.89, P < .0001).
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Table 1.
Clinical and Laboratory Characteristics of Liver
Cirrhotic Patients According to Child-Pugh's Classification
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A marked increase in Isoprostane-F2-III was detected in
cirrhotic patients, particularly in those with the highest degree of
liver failure. Thus, cirrhotic patients of C class had higher values of
Isoprostane-F2-III compared with those of B (P = .02) and A (P = .019) classes (Table 1). According to our
previous study,15 we found a significant correlation
between Isoprostane-F2-III and endotoxemia (Rho = 0.72, P < .0001). Isoprostane-F2-III was also
significantly correlated with F1+2 (Rho = 0.85, P < .0001; Fig 1A) and TF antigen (Rho = 0.95, P < .0001) and activity (Rho = 0.94, P < .0001; Fig
1B), suggesting that lipid peroxidation and clotting system activation
are closely related.
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| Fig 1.
Correlation between prothrombin fragment F1+2 and
urinary excretion of Isoprostane-F2-III in cirrhotic
patients (A) and correlation between monocyte tissue factor antigen
( ) and activity ( ) and urinary excretion of
Isoprostane-F2-III in cirrhotic patients (B).
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To further explore such a relationship, we investigated whether the
administration of vitamin E had some effect on lipid peroxidation and
clotting activation. At baseline, no difference in clinical and
laboratory characteristics was observed between the groups receiving or
not receiving vitamin E (Fig 2). After
vitamin E administration, cirrhotic patients showed significantly
higher values of plasma vitamin E compared with baseline values (median [range]: 13 [11-17] v 17 [14-29] µmol/L; P = .018). We also found a significant decrease of
Isoprostane-F2-III (median [range]: 355 [170-812]
v 240 [142-560] pg/mg creatinine; P = .008 [not shown]), monocyte TF antigen (63.6 [10-101.8] v 22.0 [10-66] pg/2 × 105 monocytes; P = .012)
activity (30 [6-50] v 15 [2-30] U/2 × 105
monocytes; P = .008), and prothrombin fragment F1+2 plasma
levels (2.05 [1.15-3.30] v 1.35 [0.90-2.20] nmol/L;
P = .008; Fig 2A and B). On the contrary, endotoxin serum
levels showed similar values before and after vitamin supplementation
(median [range]: 28.7 [3.5-62] v 29.5 [4.5-54] pg/mL;
P = .139). In patients receiving only standard treatment, no
changes in Isoprostane-F2-III, monocyte TF antigen and
activity, and F1+2 levels were observed (Fig 2A and B).
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| Fig 2.
(A) Prothrombin fragment F1+2 plasma levels and (B)
monocyte TF antigen and activity in cirrhotic patients before and after
vitamin E administration and in placebo group. *P = .012;
**P = .008. The horizontal lines refer to the median
values.
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Further support for this finding was provided by in vitro study
performed in monocytes taken from healthy subjects. In this experimental model, we tested the effect of 50 µmol/L vitamin E on
the LPS-stimulated (0.4 ng/mL) monocyte activation. The results of this
experiment are summarized in Fig 3, showing
that 50 µmol/L vitamin E significantly reduced the monocyte
expression of TF antigen (52%; P = .001) and activity
(55%; P = .003). This finding likely accounts for the
effect of 50 µmol/L vitamin E on the formation of F1+2 mediated by
LPS-stimulated monocytes. Thus, this concentration of vitamin E
significantly reduced the rate of thrombin generation (51%;
P = .025) in samples containing LPS-stimulated monocytes and
heparinized plasma (Fig 4).
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| Fig 3.
TF antigen and activity expressed by unstimulated or
LPS-stimulated (0.4 ng/mL) monocytes added with and without vitamin E
(50 µmol/L). The paired t-test was used for statistical
analysis. *P = .003; **P = .001. The top and bottom
of the box represent the standard error. The square in the middle of
the box represents the mean value. The lines extending above and below
the box refer to the standard deviation.
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| Fig 4.
Prothrombin fragment F1+2 generation observed on
unstimulated or LPS-stimulated (0.4 ng/mL) monocytes added with and
without vitamin E (50 µmol/L) and exposed to a standard heparinized
overlay milieu (see text). The paired t-test was used for
statistical analysis. *P = .025. The top and bottom of the
box represent the standard error. The square in the middle of the box
represents the mean value. The lines extending above and below the box
refer to the standard deviation.
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DISCUSSION |
This study shows for the first time that in cirrhosis lipid
peroxidation and clotting activation are related. The urinary excretion
of Isoprostane-F2-III, which is one of the most abundant
F2-isoprostanes formed under physiological conditions in
human,27 was used as marker of lipid peroxidation. Thus, this compound has been reported elevated in several pathological conditions associated with oxidant stress such as drug poisoning, cigarette smoking, coronary reperfusion, and autoimmune
disease.16,17,28,29 Consistent with our previous
report,15 we found elevated urinary excretion of
Isoprostane-F2-III in cirrhosis, particularly in
patients with severe liver failure. Such elevation should not be
considered artefactorial, ie, related to a reduced liver clearance, for
several reasons. First of all, experimental and humans' studies demonstrated that liver is poorly involved in
Isoprostane-F2-III clearance.30,31 In
addition, we demonstrated that vitamin E administration is associated
with a significant decrease in Isoprostane-F2-III, indicating that increase in this isoprostane reflects enhanced formation more than decreased clearance.15 The relationship between enhanced lipid peroxidation and clotting activation was suggested by several lines of clinical and laboratory evidences. In the
cirrhotic population, the urinary excretion of
Isoprostane-F2-III was significantly correlated with
plasma levels of prothrombin fragment F1+2 as well as with monocyte TF
antigen and activity, suggesting a cause-effect relationship between
lipid peroxidation and clotting activation. This hypothesis was
explored in an interventional study in which lipid peroxidation and
clotting activation were measured before and after vitamin E
supplementation. We observed that, after vitamin E administration, F1+2
plasma levels and the monocyte TF expression significantly decreased.
It is noteworthy that these changes were not related to endotoxemia,
which, in fact, was not modified by the antioxidant treatment. The
results of the interventional study were further corroborated by in
vitro experiments showing that 50 µmol/L vitamin E significantly
inhibited monocyte TF expression and monocyte-induced F1+2 formation.
It is noteworthy that in this experiment monocytes were stimulated with
LPS concentration close to that found in the peripheral circulation of
cirrhotic patients5 and that 50 µmol/L vitamin E reduced TF expression and F1+2 generation by 50%, on average. This is in
accordance with a previous study showing that oxidant species enhance
monocyte expression of TF and that 50 µmol/L vitamin E reduces by
approximately 50% LPS-induced monocyte TF expression.32 Our finding also supported the results of an in vivo study showing that, in subjects receiving 1,200 U/d vitamin E, monocyte function is
reduced; thus, Devaraj et al33 demonstrated that, after
vitamin E supplementation monocyte formation of oxidant species, lipid oxidation and interleukin-1 secretion were significantly decreased.
Because of the effect of vitamin E on oxygen free radical formation, it
may be postulated that the inhibition of monocyte TF expression is due
to its antioxidant property. However, further study is necessary to
analyze whether vitamin E has the same effect of other antioxidants,
which have been shown to regulate transcriptional or
posttranscriptional activation of monocyte TF
expression.13,14
Our results may have potential clinical relevance, because the
concentration we used in vitro is achievable in human blood after
vitamin E supplementation. Therefore, administration of vitamin E could
represent an interesting new approach to modulate clotting activation
and, in turn, secondary hyperfibrinolysis in this clinical setting.
This suggestion has to be confirmed in a larger controlled trial.
In conclusion, this study provides evidence that, in liver cirrhosis,
there is a relationship between lipid peroxidation and clotting
activation and suggests that lipid peroxidation may represent an
important mechanism that mediates endotoxin-induced monocyte TF
expression. The inhibition of clotting system activation by vitamin E
may open a new avenue for the treatment of clotting disturbance with
antioxidants in this clinical setting.
| |
FOOTNOTES |
Submitted June 9, 1998; accepted December 17, 1998.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Francesco Violi, MD, I Clinica Medica,
Policlinico Umberto I, Viale del Policlinico, 00185 Roma, Italy;
e-mail: violi{at}uniroma1.it.
| |
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