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Am J Physiol Regul Integr Comp Physiol 285: R463-R469, 2003. First published May 15, 2003; doi:10.1152/ajpregu.00153.2003
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INFLAMMATION, CYTOKINES, AND TEMPERATURE REGULATION

Serum but not myocardial TNF-{alpha} concentration is increased in pacing-induced heart failure in rabbits

Stephanie Aker, Sergej Belosjorow, Ina Konietzka, Alexej Duschin, Claus Martin, Gerd Heusch, and Rainer Schulz

Institute of Pathophysiology, University of Essen Medical School, 45122 Essen, Germany

Submitted 26 March 2003 ; accepted in final form 8 May 2003


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In animals and patients with severe heart failure (HF), the serum tumor necrosis factor-{alpha} (TNF-{alpha}) concentration is increased. It is, however, still controversial whether or not such increased serum TNF-{alpha} originates from the heart itself or is of peripheral origin secondary to gastrointestinal congestion and increased endotoxin concentration. We therefore now examined TNF-{alpha} in serum, myocardium, and liver of sham-operated and HF rabbits. In nine rabbits in which HF was induced by left ventricular (LV) pacing at 400 beats/min for 3 wk, LV end-diastolic diameter was increased and systolic shortening fraction (9.4 ± 1.0 vs. 28.5 ± 1.3%, echocardiography, P < 0.05) was reduced. Serum TNF-{alpha} was higher in HF than in sham-operated rabbits (240 ± 24 vs. 150 ± 22 U/ml, WEHI-cell assay, P < 0.05). In the heart, TNF-{alpha} was located mainly in the vascular endothelium (immunohistochemistry), and TNF-{alpha} protein (920 ± 160 vs. 900 ± 95 U/g) did not differ between groups. In the liver of HF rabbits, hepatocytes expressed TNF-{alpha}, and TNF-{alpha} protein was increased compared with sham-operated rabbits (2,390 ± 310 vs. 1,220 ± 135 U/g, P < 0.05) and correlated to the number of hepatic leukocytes (r = 0.85) and serum TNF-{alpha} (r = 0.69). The intestinal endotoxin concentration was 24.5 ± 1.2 vs. 17.0 ± 3.1 endotoxin units/g wet wt (P < 0.05) in HF compared with sham-operated rabbits. In this HF model, serum but not myocardial TNF-{alpha} is increased. The increased serum TNF-{alpha} originates from peripheral sources.

liver; inflammation

EXOGENOUS tumor necrosis factor-{alpha} (TNF-{alpha}) depresses ventricular function in dogs (20, 33) and rats (10), and transgenic mice overexpressing TNF-{alpha} develop dilated cardiomyopathy (12). The endogenous myocardial TNF-{alpha} concentration is increased during aortic banding (6), acute myocardial ischemia-reperfusion (22), and after myocardial infarction (25, 37) and coronary microembolization (20, 46). During ischemia, the increased myocardial TNF-{alpha} concentration is causally involved in the development of ventricular dysfunction (20) and myocardial infarction (7).

In patients with severe heart failure (5, 15, 28, 32, 38, 45, 47), particularly in those with signs of cachexia (2, 28, 31), the serum TNF-{alpha} concentration is increased, and TNF-{alpha} concentration is an independent predictor of mortality in patients with advanced heart failure (17, 21, 40). The origin of the increased serum TNF-{alpha} concentration is, however, not clearly identified yet. While in some experimental (41) and clinical (32, 38) studies, the TNF-{alpha} concentrations did not differ in paired arterial and coronary sinus blood samples, suggesting that TNF-{alpha} is of peripheral origin, in other experimental (29) and clinical (18, 26, 44) studies the myocardial TNF-{alpha} protein expression was increased. These results suggest that even though the myocardial TNF-{alpha} concentration might be increased in failing hearts, other peripheral organs must contribute to the increased serum TNF-{alpha} concentration.

Critically ill patients often suffer from congestion of gastrointestinal organs, possibly leading to elevated endotoxin levels (39), a hypothesis originally introduced by Anker and coworkers (3). Endotoxemia, in turn, is associated with an increased concentration of circulating cytokines (23, 35). Experimentally, however, no data on the endotoxin and cytokine concentrations in gastrointestinal organs have been obtained in animals with heart failure.

We therefore measured the serum, myocardial, and hepatic TNF-{alpha} concentrations and the intestinal endotoxin concentration in conscious rabbits with pacing-induced heart failure.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Experimental model. This study was approved by the bio-ethical committee of the district of Düsseldorf, Germany, and the experiments were in accordance with the guidelines of the American Physiological Society.

Instrumentation. The experiments were performed in 20 male Chinchilla bastard rabbits (specific pathogen-free, Charles River, Kisslegg, Germany) weighing 3.4 ± 0.1 kg. The animals were anesthetized initially with a subcutaneous injection of a mixture of ketamine (50 mg/kg) and xylazine (3 mg/kg). After tracheal intubation, anesthesia was maintained with 12–25 ml/h of propofol and a bolus injection of fentanyl (0.005 mg/kg) intravenously. Ventilation was maintained using a Dräger UV-2 ventilator (Dräger, Lübeck, Germany) with 70% room air-30% oxygen. Body temperature was continuously monitored, and hypothermia was prevented using a heating pad. After left thoracotomy, a pacing lead was sutured onto the apical region of the left ventricle. The pacing lead was connected to a pacemaker (Medtronic, Düsseldorf, Germany), which was implanted subcutaneously. The chest was closed in layers and evacuated with a Bülau-Drainage. The tracheal tube was removed after spontaneous breathing was ensured. The rabbits were placed on an antibiotic regimen (enrofloxacin 12 mg/kg) for 3 days, and postoperative analgesia was achieved by administration of buprenorphine (0.03 mg/kg). After instrumentation, the rabbits were allowed to recover for 7–10 days.

Experimental protocol. When the rabbits had fully recovered from surgery, heart failure was induced in nine rabbits by rapid LV pacing at a rate of 400 beats/min for 3 wk (13, 30). Heart failure was evident from clinical signs, such as ascites, and echocardiographic parameters, such as an increase in LV end-diastolic diameter and a reduction of LV systolic shortening fraction. Eleven sham-operated rabbits served as controls.

At the end of each study, rabbits were anesthetized and arterial blood samples were collected, centrifuged, and serum stored in liquid nitrogen. After euthanasia of the rabbits, two to three tissue samples were taken from the LV free wall and the liver and frozen in liquid nitrogen and stored at -70°C for later analysis. Further samples were fixed in formalin and embedded in paraffin.

Echocardiography. LV function was measured in the short axis view at baseline and after 3 wk of pacing, with the rabbits in conscious state and the pacemaker turned off for at least 60 min. Echocardiography in two-dimensional real time and M-mode acquisition (Supervision 7000, Toshiba, Neuss, Germany) was performed using a 10-MHz sector phase array transducer. Fractional shortening was assessed [(end-diastolic diameter - end-systolic diameter)/end-diastolic diameter x 100]. Additionally, LV wall thickness was measured and LV weight calculated (14). Echocardiographic determination of LV weight closely correlated to anatomic LV weight as assessed in 10 separate rabbits (echocardiographic LV weight = 1.02 x anatomic LV weight - 0.25, r = 0.72).

Histology. The extent of myocardial fibrosis was determined using Masson-Goldner staining and expressed as percentage of field of view (3 fields of 0.075 mm2 each). Apoptosis was determined using TdT-mediated dUTP nick end labeling (TUNEL) technique (In Situ Cell Death Detection Kit, La Roche Diagnostics, Mannheim, Germany), counterstaining with bisbenzimide (HOE-33342) and phalloidin (both Sigma, Taufkirchen, Germany) (20). TUNEL-positive cardiomyocyte nuclei were counted using fluorescence microscopy (Leica DMLB, Bensheim, Germany) and calculated per square millimeter. Leukocytes were quantified in hematoxylin and eosin-stained tissue sections in myocardium and liver (19). Ten randomized fields (0.193 mm2 each) were counted, and the number of leukocytes was calculated per square millimeter. For analysis of intestinal edema, a part of the small intestine (10.1 ± 0.7 g) was dried over 3 wk at 80°C. Fresh and dry weights were measured, and the percent change was calculated.

TNF-{alpha} concentration (WEHI cell assay). Serum, myocardial, and hepatic TNF-{alpha} concentrations were determined using a cytotoxic activity assay (WEHI cells) (8, 20, 46). The TNF-{alpha} concentration was expressed in units per milliliter for serum and in units per gram for myocardium and liver (1 U is the reciprocal of the dilution necessary to cause 50% cell destruction) (8). The WEHI cell assay was calibrated using purified rabbit TNF-{alpha} (Pharmingen, San Diego, CA), which was added at given concentrations to the WEHI cell assay. The regression analysis was as follows: rabbit TNF-{alpha} = 0.0564 x WEHI cell assay + 2.7419, n = 66, r = 0.99, P < 0.001. Using the WEHI cell assay, the detection limit was ~3 pg/ml rabbit TNF-{alpha}, which is far below the detection limit of the TNF-{alpha} ELISA [~25 pg/ml (4)].

Immunohistochemistry. Paraffin-embedded specimens from heart and liver were cut into 4-µm sections, dewaxed and rehydrated with graded alcohol, and rinsed in PBS. Sections were pretreated with pepsin for antigen retrieval and then incubated with a mouse monoclonal anti-TNF-{alpha} antibody (Santa Cruz sc-7317, Santa Cruz, CA) for 1 h at 37°C diluted in 1:10 antibody diluent (Dako Kopenhagen, Denmark). Several rinsing steps with PBS followed. A secondary FITC-conjugated goat anti-mouse antibody (sc-2010) (1:100, 37°C for 1 h) was added. Negative controls included omission of primary antibodies. The samples were cover-slipped in Vectashield (H-1000, Vector Laboratories, Burlingame, CA) and examined by laser scan microscopy at x630 magnification (Zeiss LSM, Oberkochem, Germany).

Myocardial TNF-{alpha} receptors 1 and 2. Cytosolic proteins were prepared using a modification of the method described by Schreiber et al. (43). The myocardial samples were weighed, diluted with buffer [1:10, 20 mM Tris·HCl, 0.33 M sucrose, 5 mM EDTA, 0.5 mM EGTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 0.005% leupeptin; pH 7.4], and homogenized. The homogenates were then centrifuged for 20 min at 20,000 g. The supernatant was saved for cytosolic protein analysis. For preparation of membrane extracts the pellet was rehomogenized with buffer (1:5, 20 mM Tris·HCl, 0.33 M sucrose, 5 mM EDTA, 0.5 mM EGTA, 1 mM PMSF, 0.005% leupeptin, and 1% Triton X-100; pH 7.4), incubated on ice for 30 min, and centrifuged for 45 min at 20,000 g. The supernatant was saved for membrane protein analysis. Protein concentrations were determined using the bicinchoninic acid protein assay (Pierce, Rockford, IL).

Cytosolic (60 µg) and membrane (50 µg) proteins were loaded on 10% PAGE-SDS gels. The proteins were separated by electrophoresis (25 µA, for 2 h at 4°C), and the separated proteins were transferred to nitrocellulose membranes by electroblotting (40 V, overnight at 4°C). The membranes were incubated in Tris-buffered saline (TBS; 20 mM Tris and 120 mM NaCl at 25°C) containing 5% nonfat dry milk for 90 min followed by incubation with either specific polyclonal anti-TNF receptor 1 or specific polyclonal anti-TNF receptor 2 antibodies (StressGen Biotechnologies, Victoria, BC, Canada) overnight at 4°C. The blots were then washed four times with TBS containing 0.05% Tween 20 (TTBS) and were then incubated for 1 h with a secondary antibody (anti-rabbit immoglobulin linked to horseradish peroxidase; Santa Cruz). After four washes with TTBS, detection was performed by enhanced chemiluminescence. The resulting autoradio-graphs were analyzed by quantitative two-dimensional densitometry using commercially available software (Herolab, Wiesloch, Germany). The two-dimensional band intensity of cytosolic and membrane TNF receptors was expressed as percentage of the total TNF receptor content.

Endotoxin concentrations. Arterial blood samples were collected in sterile, endotoxin-free tubes and diluted with LAL Reagent Water (1:1). Tissue samples (intestine and liver) were weighed, collected in sterile, endotoxin-free tubes, diluted with LAL Reagent Water (1:10), and homogenized. The samples were centrifuged at 14,000 g for 10 min, and the supernatants were collected for the measurements of endotoxin. Inactivation of inhibitors was obtained by heating all samples for 15 min at 75°C. A chromogenic kinetic limulus amebocyte lysate assay (LAL assay QCL-1000, BioWhit-taker, Walkersville, MD) was used to measure the endotoxin concentration (9). The concentration of endotoxin was calculated from a standard curve (Esherichia coli endotoxin 0111: B4) and expressed in endotoxin units (EU) per milliliter for serum and EU per gram for intestinal and hepatic tissues.

Statistical analysis. Values are expressed as means ± SE. Statistical comparison of all data before and after 3 wk of pacing between the two groups was performed by two-way ANOVA. When a significant overall effect was detected, individual mean values were compared using Bonferroni's method. TNF-{alpha} concentration, TNF-{alpha} receptors 1 and 2, extent of fibrosis, and number of TUNEL-positive cells and leukocytes were compared between sham-operated and heart failure rabbits using an unpaired t-test. Statistical significance was accepted for a P value <0.05.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Hemodynamics. Heart rate did not change significantly from baseline to 3 wk in sham-operated (260 ± 8 to 236 ± 9 beats/min) and heart failure rabbits (265 ± 15 to 260 ± 14 beats/min) and also did not differ between groups. Heart failure was characterized by an increase of LV end-diastolic diameter from 14.7 ± 0.6 to 18.4 ± 0.6 mm and a reduction of LV systolic shortening fraction from 32.6 ± 1.5 to 9.4 ± 1.0% (both P < 0.05) from baseline to 3 wk of pacing. In the sham-operated group, LV end-diastolic diameter (14.9 ± 0.5 to 15.2 ± 0.3 mm) and LV systolic shortening fraction (30.1 ± 1.4 to 28.5 ± 1.3%) remained unchanged over the time course of the protocol. There was no difference in LV weight from baseline to 3 wk in sham-operated (5.1 ± 0.5 vs. 5.4 ± 0.3 g) and heart failure rabbits (5.0 ± 0.4 vs. 5.3 ± 0.4 g). Histology. The extent of fibrosis (31.4 ± 1.8 vs. 7.0 ± 0.8% of analyzed area, P < 0.05, Fig. 1) and the number of TUNEL-positive cardiomyocyte nuclei (0.033 ± 0.004 vs. 0.002 ± 0.001/mm2, P < 0.05) was greater in failing than in sham-operated hearts. The number of leukocytes in myocardium was not different between failing and sham-operated hearts (174 ± 20 vs. 142 ± 9 cells/mm2). In the liver, the number of leukocytes was significantly greater in heart failure than in sham-operated rabbits (108 ± 13 vs. 52 ± 3 cells/mm2, P < 0.05, Fig. 1). The percentage of dry vs. fresh small intestinal weight was not different between groups (19.0 ± 1.1 vs. 18.2 ± 0.8%).



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  		Fig. 1. Myocardial fibrosis (Masson-Goldner trichrome staining) in a heart failure (A) and a sham-operated (B) rabbit. Scale bar, 50 µm. Liver (hematoxylin and eosin staining) in a heart failure (C) and a sham-operated (D) rabbit. Scale bar, 50 µm.

 

TNF-{alpha} concentration (WEHI cell assay). Serum TNF-{alpha} concentration (240 ± 24 vs. 150 ± 22 U/ml, P < 0.05) and hepatic TNF-{alpha} concentration (2,390 ± 310 vs. 1,220 ± 135 U/g, P < 0.05) were increased in heart failure compared with sham-operated rabbits (Fig. 2). In contrast, the myocardial TNF-{alpha} concentration (920 ± 160 vs. 900 ± 95 U/g, Fig. 2) did not differ between groups and did not correlate to the serum TNF-{alpha} concentration (Fig. 3A). In contrast, both the hepatic TNF-{alpha} concentration and the number of leukocytes (Fig. 3B) and the serum and hepatic TNF-{alpha} concentrations (Fig. 3C) were correlated.



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  		Fig. 2. Tumor necrosis factor-{alpha} (TNF-{alpha}) concentrations in serum, myocardium, and liver of heart failure and sham-operated rabbits. TNF-{alpha} concentration in serum is expressed as U/ml, whereas TNF-{alpha} concentrations in myocardium and liver are expressed as U/g wet wt. Values are means ± SE. *P < 0.05 vs. sham operated.

 


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  		Fig. 3. A: correlation between the myocardial and serum TNF-{alpha} concentrations. B: correlation between the hepatic leukocyte count and the hepatic TNF-{alpha} concentration. C: correlation between the hepatic and the serum TNF-{alpha} concentrations.

 

Immunohistochemistry. In the heart, TNF-{alpha} was almost entirely localized in the vascular endothelium in heart failure and sham-operated rabbits. In the liver of sham-operated rabbits, TNF-{alpha} was again localized in the vascular endothelium; however, in heart failure rabbits, TNF-{alpha} was also expressed in hepatocytes (Fig. 4).



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  		Fig. 4. TNF-{alpha} localization in heart and liver (immunohistochemistry) in a sham-operated (A, C) and in a heart failure (B, D) rabbit.

 

TNF-{alpha} receptors 1 and 2. Both the cytosolic [333,772 ± 45,024 vs. 293,878 ± 27,858 arbitrary units (AU)] and the membrane protein concentrations (692,434 ± 93,926 vs. 575,047 ± 41,959 AU) of TNF-{alpha} receptor 1 did not differ between heart failure and sham-operated rabbits. Also, the cytosolic (99,731 ± 12,868 vs. 97,498 ± 5,825 AU) and the membrane protein concentrations (188,016 ± 26,768 vs. 201,297 ± 15,587 AU) of TNF-{alpha} receptor 2 were similar between groups.

Endotoxin concentration. The intestinal endotoxin concentration was increased in heart failure compared with sham-operated rabbits (24.5 ± 1.2 vs. 17.0 ± 3.1 EU/g wet wt, P < 0.05). Serum and hepatic endotoxin concentrations did not differ between heart failure (1.7 ± 0.3 EU/ml, 66.7 ± 11.3 EU/g wet wt, respectively) and sham-operated rabbits (1.6 ± 0.2 EU/ml, 73.4 ± 7.9 EU/g wet wt, respectively).


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The main finding of the present study is that the increased serum TNF-{alpha} is of peripheral origin and not derived from the myocardium in this heart failure model.

About one-half of all patients with severe heart failure have increased serum TNF-{alpha} concentration (5, 15, 28, 32, 38, 45, 47). Particularly in those with signs of cachexia (2, 28, 31), the serum TNF-{alpha} concentration is increased, and the serum TNF-{alpha} concentration is an independent predictor of mortality in patients with advanced heart failure (17, 21, 40).

In the present study, we as others before (6, 25) could detect TNF-{alpha} within the heart mainly localized to the vascular endothelium. However, the myocardial TNF-{alpha} protein concentration was not elevated in failing hearts, suggesting that the increased serum TNF-{alpha} concentration was not of cardiac origin.

The finding of an unchanged myocardial TNF-{alpha} concentration in the pacing-induced heart failure model, in which baseline transmural blood flow is only minimally affected and signs of ischemia are absent (34), contrasts with results obtained in models of LV dysfunction secondary to aortic banding (6), coronary microembolization (20, 46), or myocardial infarction (25, 37). During ischemia, the myocardial TNF-{alpha} concentration is rapidly increased within the area at risk (22, 24, 25). With prolongation of ischemia and development of cardiomyocyte necrosis, the TNF-{alpha} concentration increases also in the surrounding viable portions of the myocardium (20, 37, 46). Indeed, in the scenario of myocardial ischemia-reperfusion, treatment with TNF-{alpha} antibodies reduced the extent of myocardial infarction (7) and attenuated the contractile dysfunction after coronary microembolization (20, 46). In the latter studies, the serum TNF-{alpha} concentration remained unaltered, thereby supporting the notion of a direct action of TNF-{alpha} on the level of the cardiomyocytes during ischemia-reperfusion.

In heart failure, the increased serum TNF-{alpha} concentration could result from the peripheral congestion and potential endotoxemia (3, 39) rather than being released from the heart itself. Indeed, endotoxin when administered to whole blood samples taken from heart failure patients increased the serum TNF-{alpha} concentration (23), indicating that blood constituents could produce or release TNF-{alpha}. Further supporting the view that TNF-{alpha} is of peripheral origin, the TNF-{alpha} concentration in the arterial and coronary sinus blood remained unchanged in dogs with pacing-induced heart failure (41) and in patients with severe heart failure (32, 38), although the serum TNF-{alpha} concentration was increased.

Although congestion of the small intestine was not detected in the present study, the endotoxin concentration of the small intestine was increased in heart failure compared with sham-operated rabbits. In contrast, serum and hepatic endotoxin concentrations were comparable between groups. Two potential explanations for the increased serum TNF-{alpha} concentration could be envisaged: first, the liver clears endotoxin from the intestinal blood draining into the portal vein. Such increased endotoxin clearance is then associated with increased hepatic leukocyte infiltration and TNF-{alpha} concentration in heart failure rabbits. Indeed, in heart failure rabbits TNF-{alpha} was expressed in hepatocytes (Fig. 4). The increased hepatic TNF-{alpha} concentration correlated to the increased serum TNF-{alpha} concentration (Fig. 3C). Second, the increased endotoxin concentration in the intestinal blood might stimulate TNF-{alpha} production or release from blood constituents (23); such TNF-{alpha} is subsequently cleared in part by the liver. The increased hepatic TNF-{alpha} concentration then causes an increase in the hepatic leukocyte concentration, as recently demonstrated in rats in vivo (48). In the present study, however, we could not distinguish between these two possibilities.

One explanation for the progression of LV dysfunction in heart failure relates to an increased level of apoptosis (1, 36). Also, in the present study the extent of apoptosis was increased in failing hearts. Although an increased TNF-{alpha} concentration induces apoptosis in skeletal muscle (16), the primarily vascular localization of TNF-{alpha} and the unchanged myocardial TNF-{alpha} concentration in the present study do not support the idea that TNF-{alpha}-induced apoptosis contributed to the induction or progression of LV dysfunction in this heart failure model. Similarly, in explanted failing human hearts cardiomyocyte apoptosis was increased (0.041 vs. 0.007%) without a correlation to the highly variable TNF-{alpha} concentration (42).

In mice lacking the myocardial TNF-{alpha} receptors 1 and 2, the ischemia-induced cardiomyocyte apoptosis was increased (27). Thus a decrease of the myocardial TNF-{alpha} receptor density, in the presence of an unchanged myocardial TNF-{alpha} concentration, could partially be responsible for the increased number of apoptotic cardiomyocytes observed in failing hearts. Indeed, a reduction in the density of TNF-{alpha} receptors 1 and 2 was measured in explanted failing human hearts (47). In the present study, however, the myocardial TNF-{alpha} receptor density was not different between failing and sham-operated hearts, thus excluding an important role of TNF-{alpha} and its receptors for the observed morphological alterations in the failing heart.

From the results of the present study, we can rule out a direct action of TNF-{alpha} on the level of the cardiomyocytes. However, the increased circulating TNF-{alpha} concentration might still contribute to the progression of heart failure. Circulating TNF-{alpha} activates angiotensinogen gene expression in hepatocytes (11), thereby potentially contributing to the increased circulating ANG II concentration measured in heart failure, and also activates neurons in the paraventricular nucleus of the hypothalamus, leading to an increased cardiac sympathetic nerve activity (49), thereby potentially contributing to the increased myocardial norepinephrine concentration measured in heart failure. Thus, despite an unchanged myocardial TNF-{alpha} concentration, the increased circulating TNF-{alpha} concentration might contribute to the depression of LV function during the progression of heart failure.

Summarizing the data, the changes in the serum and myocardial TNF-{alpha} concentrations in heart failure appear to be independent from each other. The increased serum TNF-{alpha} concentration is of peripheral origin and most likely secondary to LV dysfunction.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. Schulz, Institute of Pathophysiology, Univ. of Essen Medical School, Hufelandstraße 55, 45122 Essen, Germany (E-mail: rainer_schulz{at}uni-essen.de).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 DISCUSSION
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