Adipose Tissue Interleukin-18 mRNA and Plasma Interleukin-18: Effect of Obesity and Exercise

Authors

  • Lotte Leick,

    Corresponding author
    1. Centre of Inflammation and Metabolism, University of Copenhagen, Copenhagen, Denmark
    2. Copenhagen Muscle Research Centre, University of Copenhagen, Copenhagen, Denmark
    3. Department of Molecular Biology, The August Krogh Building, University of Copenhagen, Copenhagen, Denmark
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  • Birgitte Lindegaard,

    1. Centre of Inflammation and Metabolism, University of Copenhagen, Copenhagen, Denmark
    2. The Department of Infectious Diseases, Rigshospitalet
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  • Dorthe Stensvold,

    1. Centre of Inflammation and Metabolism, University of Copenhagen, Copenhagen, Denmark
    2. Copenhagen Muscle Research Centre, University of Copenhagen, Copenhagen, Denmark
    3. Department of Molecular Biology, The August Krogh Building, University of Copenhagen, Copenhagen, Denmark
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  • Peter Plomgaard,

    1. Centre of Inflammation and Metabolism, University of Copenhagen, Copenhagen, Denmark
    2. Copenhagen Muscle Research Centre, University of Copenhagen, Copenhagen, Denmark
    3. The Department of Infectious Diseases, Rigshospitalet
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  • Bengt Saltin,

    1. Copenhagen Muscle Research Centre, University of Copenhagen, Copenhagen, Denmark
    2. Rigshospitalet, Copenhagen, Denmark.
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  • Henriette Pilegaard

    1. Centre of Inflammation and Metabolism, University of Copenhagen, Copenhagen, Denmark
    2. Copenhagen Muscle Research Centre, University of Copenhagen, Copenhagen, Denmark
    3. Department of Molecular Biology, The August Krogh Building, University of Copenhagen, Copenhagen, Denmark
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August Krogh Building, Universitetsparken 13, 2100 Copenhagen, Denmark. E-mail: LLeick@aki.ku.dk

Abstract

Objectives: Obesity and a physically inactive lifestyle are associated with increased risk of developing insulin resistance. The hypothesis that obesity is associated with increased adipose tissue (AT) interleukin (IL)-18 mRNA expression and that AT IL-18 mRNA expression is related to insulin resistance was tested. Furthermore, we speculated that acute exercise and exercise training would regulate AT IL-18 mRNA expression.

Research Methods and Procedures: Non-obese subjects with BMI < 30 kg/m2 (women: n = 18; men; n = 11) and obese subjects with BMI >30 kg/m2 (women: n = 6; men: n = 7) participated in the study. Blood samples and abdominal subcutaneous AT biopsies were obtained at rest, immediately after an acute exercise bout, and at 2 hours or 10 hours of recovery. After 8 weeks of exercise training of the obese group, sampling was repeated 48 hours after the last training session.

Results: AT IL-18 mRNA content and plasma IL-18 concentration were higher (p < 0.05) in the obese group than in the non-obese group. AT IL-18 mRNA content and plasma IL-18 concentration was positively correlated (p < 0.05) with insulin resistance. While acute exercise did not affect IL-18 mRNA expression at the studied time-points, exercise training reduced AT IL-18 mRNA content by 20% in both sexes.

Discussion: Because obesity and insulin resistance were associated with elevated AT IL-18 mRNA and plasma IL-18 levels, the training-induced lowering of AT IL-18 mRNA content may contribute to the beneficial effects of regular physical activity with improved insulin sensitivity.

Introduction

Obesity and a physically inactive lifestyle are associated with an increased risk for developing insulin resistance and atherosclerosis. Obesity is recognized to be associated with elevated circulating levels of both interleukin (IL)1 (1) and tumor necrosis factor α (TNF-α) (1, 2, 3, 4), and elevated plasma levels of some inflammatory markers may reflect obesity rather than insulin resistance (5, 6). Adipose tissue (AT) has been characterized as an endocrine organ and an important source of cytokines including IL-6 (7) and TNF-α (8), and it has been suggested that increased production and secretion of proinflammatory cytokines by AT may play a role in development of insulin resistance.

Contrasting the accumulating data on the multiple metabolic roles of TNF-α and IL-6 (9), the metabolic role of IL-18 has not been studied to the same extent, although elevated systemic IL-18 levels have been associated with a number of diseases. Thus, an enhanced plasma IL-18 concentration predicts cardiovascular mortality in patients with coronary atherosclerosis (10), and a role for IL-18 in the autoimmune β-cell destruction leading to type 1 diabetes has been proposed (11, 12, 13). Importantly, recent studies also reported that plasma IL-18 levels are elevated both in patients with type 2 diabetes (14, 15, 16, 17) and in obese individuals (18, 19).

IL-18 mRNA is expressed in human AT (20), and both AT IL-18 mRNA content and systemic levels of IL-18 show a relationship with low limb fat content and high waist-to-hip ratio (WHR) in patients with HIV-coupled lipodystrophy, who are characterized by severe fat redistribution with peripheral fat loss and visceral fat accumulation (20, 21).

Because the above-mentioned diseases are all linked with insulin resistance, it may be speculated that circulating IL-18 is an inflammatory marker of insulin resistance in addition to being related to obesity. In accordance with this idea is that, in addition to plasma IL-18 concentrations being increased in obese women and decreased after weight loss, plasma IL-18 concentrations have been shown to correlate with surrogate indices of insulin resistance, such as the homeostasis model assessment (HOMA-IR), WHR, and fasting insulin levels (16, 17, 21). Therefore, IL-18 may indeed be a useful marker of the inflammatory process associated with both obesity and insulin resistance.

Regular exercise offers protection against atherosclerosis, type 2 diabetes, colon cancer, and breast cancer (22), which are diseases accompanied by low-grade systemic inflammation (9). Cross-sectional studies have shown an association between physical inactivity and low-grade systemic inflammation in healthy subjects, in elderly people, and in patients with intermittent claudication, and longitudinal studies have shown that regular exercise induces a reduction in C-reactive protein levels (22). Furthermore, acute exercise was shown to inhibit endotoxin-induced enhancement of plasma TNF-α (23), and this finding was supported by a recent study showing that exercise normalizes overexpression of TNF-α in TNF receptor knockout mice (24). Together these observations support that regular physical activity may suppress systemic low-grade inflammation (9, 23, 25).

The aim of this study was to test the hypothesis that obesity and insulin resistance are associated with elevated AT IL-18 mRNA expression and plasma IL-18 levels. We further hypothesized that acute exercise would regulate AT IL-18 mRNA and that regular exercise training would decrease both AT IL-18 mRNA expression and systemic levels of IL-18 in obese individuals.

Research Methods and Procedures

Subjects

Non-obese subjects with BMI <30 kg/m2 (18 women and 11 men), with an average age of 25.5 ± 1.1 (women) and 25.6 ± 1.2 [standard error (SE)] years (men) and a BMI of 25.1 ± 0.8 (women) and 24.1 ± 1 kg/m2 (men), and obese subjects with a BMI >30 kg/m2 (6 women and 7 men), with an average age of 34.5 ± 3.6 (women) and 37.1 ± 3.7 years (men) and a BMI of 38.5 ± 3.6 (women) and 32.5 ± 0.6 kg/m2 (men), participated in the study.

All subjects were healthy and untrained. The subjects were fully informed of the nature and possible risks associated with the study before they gave their consent. The study was approved by the Copenhagen and Frederiksberg Ethics Committee, and the experiments conformed to the Declaration of Helsinki.

Experimental Protocols

Approximately 1 week before the experimental day, fasting blood samples were drawn, and whole body maximal oxygen uptake (Vo2max) was determined using a standard progressive exercise test on a bicycle ergometer.

The subjects were asked to refrain from vigorous physical activity and instructed to eat a standardized diet for 48 hours before the experimental day. On the experimental day, subjects arrived at the laboratory 2 hours after a light standardized meal, an AT biopsy was obtained from the subcutaneous abdominal AT, and a blood sample was drawn using a catheter placed in an antecubital arm vein. From that point, the subjects went through three different protocols (A–C).

Protocol A

non-obese female subjects (n = 10) performed a 2-hour exercise bout at 60% of Vo2max using a bicycle ergometer. The exercise bout was followed by additional AT biopsies from the subcutaneous abdominal AT just after exercise (0 hours) and after 2 hours of recovery from exercise (2 hours). A control study including eight non-obese female subjects was performed with AT biopsies and blood samples taken at the same time-points but with no exercise bout.

Protocol B

non-obese male subjects (n = 11) performed a 1.5-hour exercise bout at 70% of Vo2max using a bicycle ergometer. Afterward, they received a light snack. An additional AT biopsy was taken after 10 hours of recovery. Additionally, all subjects completed a control trial with the same procedure but without the exercise bout.

Protocol C

Obese subjects (seven men and six women) completed a supervised 8-week high-intensity training program, three times per week, 30 min/d, performed on a rowing ergometer. The exercise intensity was initially ∼70% Vo2max, and the resistance was continually increased during the training period as performance/endurance was improved. After the training period, AT biopsies and blood samples were obtained 48 hours after the last training session. One woman did not complete the exercise trial.

AT Biopsies

The AT biopsies were obtained using the percutaneous needle biopsy technique with suction. The AT biopsies were obtained in separate incisions or with the needle pointing in opposite direction of the prior biopsy. Each AT sample was quickly dipped on gauze to remove superficial blood, and with the tissue on a pre-cooled aluminum block, blood and connective tissue were removed. The samples were quickly frozen in liquid N2 and stored at −80 °C for later RNA isolation.

Blood Sample Analyses

Samples for blood analyses were immediately transferred to tubes containing EDTA as an anticoagulant and centrifuged at 2465g for 15 minutes. Plasma concentrations of total cholesterol, free fatty acids (FFAs), triglycerides (TGs), and glucose were measured using an automatic spectrophometer (Cobas FARA 2; Roche Diagnostics, Basal, Switzerland). Plasma insulin and plasma IL-18 were determined by enzyme linked immunosorbent assay kits (insulin: DakoCytomation, Glostrup, Denmark; IL-18: MBL Medical and Biological Laboratories, Nagoya, Japan).

RNA Isolation

AT RNA isolation was performed using TRIzol reagent (Invitrogen, Carlsbad, CA). Pieces of 70 to 80 mg of AT were homogenized in 2 mL Trizol for 20 seconds using a Kinematic Polytron PT2100 (Kinematic, Luzern, Switzerland) following the procedure provided by the manufacturer (Invitrogen). The final pellet was resuspended in di-ethyl pyrocarbonate-treated water (1 μL/mg original tissue weight) containing 0.1 mM EDTA and stored at −80 °C.

Reverse Transcription and Polymerase Chain Reaction

Reverse transcription (RT) was performed using the superscript II RNase H-system from Invitrogen as previously described (26). RT products were diluted in nuclease-free H2O with each sample originating from 11 μL of total RNA diluted to a total volume of 100 μL.

IL-18 mRNA expression was determined by real-time polymerase chain reaction (PCR; ABI PRISM 7900 Sequence Detection System; Applied Bio systems, Foster City, CA) using a pre-developed assay reagent (Applied Biosystems) as previously described (27).

Determination of Single-stranded DNA Content

The IL-18 mRNA content was normalized to the total single-stranded DNA content in each RT sample measured by a fluorescence-based method using Oligreen reagent (Molecular Probes, Leiden, The Netherlands) as previously described (28).

Statistics and Calculations

Using fasting plasma concentrations of glucose and insulin, the level of insulin resistance was calculated by the HOMA version 1996 (available at www.dtu.ox.ac.uk) as the original HOMA described by Matthews in 1985 tends to overestimate insulin resistance (29, 30).

Before statistical analysis, parameters were log-transformed to approximate a normal distribution. Statistical calculations were performed using Sigma stat statistical software (version 2.03; Chicago, IL). Student's t test was used for comparison of anthropometric and clinical data between non-obese and obese subjects and between sexes. Paired Student's t test was used for comparison between anthropometric data and clinical data in obese subjects before and after 8 weeks of training. The impact of BMI and sex on IL-18 mRNA expression and plasma IL-18 concentration was tested by two-way ANOVA, as was the impact of acute exercise/control on IL-18 mRNA expression. The effect of training and sex was evaluated by two-way ANOVA for repeated measurements. The Student-Newman-Keul post hoc test was used to locate differences. Regression analysis between HOMA-IR and age with AT IL-18 mRNA expression and AT IL-18 plasma concentration was performed using Pearson correlation index. p < 0.05 was considered significant, and a tendency was reported when 0.05 < p < 0.10 in all analyses. All results are presented as mean ± SE.

Results

Anthropometric and Clinical Data

Fasting plasma insulin was 30% to 50% higher (p < 0.05) in the obese subjects than in the non-obese subjects within each sex. Within obese female subjects, HOMA-IR, plasma FFAs, and plasma cholesterol was 30% to 50% higher (p < 0.05) than non-obese female subjects. In addition, men within the obese group tended (p < 0.10) to have 10% to 30% higher HOMA-IR and plasma cholesterol than nonobese men (Table 1).

Table 1.  Anthropometric and clinical characteristics of the subjects
 non-obese BMI < 30Obese BMI ≥ 30Obese, BMI ≥ 30 after training
 Women (n = 18)Men (n = 11)Women (n = 6)Men (n = 7)Women (n = 5)Men (n = 7)
  • Vo2max, maximal oxygen uptake; HOMA-IR, insulin sensitivity as measured by the homeostasis model assessment; FFA, free fatty acid; TG, triglyceride.

  • *

    Significantly different from non-obese within gender.

  • Males significantly different from females within non-obese or obese group.

  • Tendency for difference from non-obese subjects within gender.

  • §

    Significantly different from before training (data from males and females are pooled).

  • n= 8–10.

Vo2max (LO2/min)2.55 ± 0.163.76 ± 0.182.4 ± 0.143.5 ± 0.172.59 ± 0.20§3.79 ± 0.17§
Insulin (Pmol/L)41.1 ± 7.216.3 ± 2.663.4 ± 6*36.1 ± 7.3*69 ± 1134.9 ± 8
Glucose (mmol/L)4.7 ± 0.14.9 ± 0.15.0 ± 0.35.2 ± 0.25.0 ± 0.45.1 ± 0.2
HOMA–IR0.7 ± 0.10.5 ± 0.11.2 ± 0.1*0.7 ± 0.11.3 ± 0.20.7 ± 0.1
FFA (μmol/L)600 ± 51619 ± 58903 ± 132*543 ± 86904 ± 152645 ± 87
Cholesterol (mmol/L)4.3 ± 0.24.3 ± 0.36.4 ± 0.4*5.0 ± 0.25.9 ± 0.95 ± 0.4
TG (μmol/L)959 ± 79945 ± 781007 ± 2141271 ± 2021134 ± 3331295 ± 410

Within each sex, the obese subjects were older (p < 0.05) than the non-obese group. Within the obese and nonobese groups, men had ∼30% higher (p < 0.05) Vo2max and 30% to 60% lower (p < 0.05) fasting insulin concentrations and HOMA-IR than women within the same group. In addition, within the obese group, men had 20% to 60% lower (p < 0.05) BMI, plasma cholesterol, and plasma FFAs than women (Table 1).

After exercise training for 8 weeks, men within the obese group had 15% to 50% lower (p < 0.05) BMI, fasting insulin, HOMA-IR, plasma FFAs, and plasma cholesterol than obese women. Exercise training of the obese subjects for 8 weeks induced an 8% increase in Vo2max (p < 0.05; Table 1).

IL-18 mRNA Expression in AT and Plasma Content

AT IL-18 mRNA content was elevated (p < 0.05) by ∼80% in obese men and by ∼130% in obese women compared with the non-obese group (Figure 1A). The plasma IL-18 concentration was ∼35% higher (p < 0.05) in the obese men and ∼40% higher in obese women than the non-obese subjects (Figure 1B). There were no sex-specific differences in either AT IL-18 mRNA content or plasma IL-18 concentration (Figure 1A and B). AT IL-18 mRNA content and plasma IL-18 concentration were significantly positively correlated with the surrogate measure of insulin resistance HOMA-IR in both women (AT IL-18 mRNA content/plasma IL-18 concentration: r2 = 0.66; p < 0.001/r2 = 0.28, p < 0.05) and men (AT IL-18 mRNA content/plasma IL-18 concentration: r2 = 0.36; p < 0.05/r2 = 0.43, p < 0.05; Figure 2A and B). In addition, AT IL-18 mRNA content was significantly positively correlated with plasma IL-18 concentration in women (r2 = 0.28; p < 0.05) and men (r2 = 0. 46; p < 0.05; data not shown).

Figure 1.

(A) AT IL-18 mRNA expression in non-obese subjects with BMI <30 kg/m2 (women: n = 18; men: n = 11) and obese subjects with BMI ≥30 kg/m2 (women: n = 6; men: n = 7). Data are presented as the ratio between IL-18 mRNA and single-stranded DNA content. (B) Venous plasma (IL-18) concentration (picograms per milliliter) in non-obese subjects with BMI <30 kg/m2 (women: n = 10; men: n = 8) and obese subjects with BMI ≥30 kg/m2 (women: n = 6; men: n = 5). Data are presented as the ratio between IL-18 mRNA and single-stranded DNA content. Values are mean ± SE. *Differences between non-obese and obese, p < 0.05.

Figure 2.

Correlation analysis of (A) AT IL-18 mRNA expression given as the ratio between IL-18 mRNA and single-stranded DNA content and HOMA-IR in male and female subjects and (B) venous plasma IL-18 concentration (picograms per milliliter) and HOMA-IR in male and female subjects.

Effects of Acute Exercise and Training

A prolonged exercise bout did not induce changes in AT IL-18 mRNA expression either immediately after exercise or at 2-hour of recovery in non-obese women (Figure 3A) or at 10-hour of recovery in non-obese men (Figure 3B).

Figure 3.

AT IL-18 mRNA response during recovery from exercise. (A) Non-obese female subjects with BMI <30 kg/m2 (n = 10) completed a 2-hour bicycle exercise bout at 60% of Vo2max, and AT biopsies were obtained from the abdominal AT before (Pre), just after exercise (0 hours), and after 2 hours of recovery (2 hours). A control study including eight non-obese female subjects was performed with AT biopsies taken at the same time-points but with no exercise bout. (B) Non-obese men with BMI <30 kg/m2 (n = 11) completed a 1.5-hour bicycle exercise bout at 70% of Vo2max, and AT biopsies were obtained from the abdominal AT before (Pre) and after 10-hour of recovery. Additionally, all subjects completed a control trial with the same procedure only without the exercise. IL-18 mRNA is given as the ratio between IL-18 mRNA and single-stranded DNA content. Data are expressed relative to the Pre level, which was set to 1.0. Values are means ± SE.

To examine whether exercise training could lower the elevated AT IL-18 mRNA and plasma IL-18 levels in obese individuals, seven male and five female subjects of the obese group underwent an 8-week training program. Training resulted in a reduced (p < 0.05) AT IL-18 mRNA content when data from men and women were pooled and a tendency for ∼20% lower (p = 0.06) IL-18 AT mRNA content in both male and females subjects when analyzed separately (Figure 4A). Although the exercise training induced a reduction in plasma IL-18 concentration by 14% in men and 25% in women, this was not significant (p = 0.16; Figure 4B).

Figure 4.

Effect of training on AT IL-18 mRNA content and plasma IL-18 concentration. (A) AT IL-18 mRNA content and (B) fasting venous plasma IL-18 concentration (picograms per milliliter) before and after 8 weeks of training in male and female obese subjects. Abdominal AT and venous plasma samples were obtained 48 hours after the last training session. IL-18 mRNA content is given as the ratio between IL-18 mRNA content and single-stranded DNA. Values are mean ± SE, n = 5–7. § Significant difference between before and after training when pooling male and female data, p = 0.06.

Discussion

The major findings of this study are that obese individuals had higher IL-18 mRNA content in the abdominal adipose tissue than non-obese subjects and that exercise training lowered the elevated IL-18 mRNA levels in obese subjects. In addition, IL-18 mRNA and plasma IL-18 correlated with insulin resistance, suggesting that training-induced reduction in AT IL-18 expression may be a contributing mechanism to improve insulin sensitivity after training. There were no sex-specific differences in either the AT IL-18 mRNA expression or fasting plasma IL-18.

This study adds to previous findings showing an association between plasma IL-18 levels and obesity (19, 31, 32) and suggesting a relationship between plasma IL-18 concentrations and insulin resistance (16, 17, 19). The observed association between AT IL-18 mRNA content and insulin resistance supports the hypothesis that insulin resistance is associated with an elevated production of IL-18 (16, 33) and that AT could be an important source of IL-18 (31, 34). The recent findings that low plasma IL-18 correlated with both weight loss and decreased WHR (31, 32), and that isolated human adipocytes secrete IL-18, are in line with the idea that AT may be a source of circulating IL-18 (34) and that increased levels of inflammatory proteins in plasma may partly reflect a spillover from the AT, potentially leading to systemic inflammation. These observations thus support the hypothesis that AT acts as an endocrine organ, producing a number of proteins referred to as the adipokines, which are thought to play important endocrine roles and to be involved in obesity-associated complications (9, 35).

It should be mentioned that the obese subjects in the present study were older than the non-obese subjects, and because plasma inflammation has been shown to increase in elderly people (36), it may be argued that the age difference contributed to the IL-18 difference. However, the subjects in this study are well below the age where age-related inflammatory processes are evident.

The possibility that lifestyle changes can evoke alterations in adipokine mRNA expression was recently shown in severely obese subjects accomplishing reduced IL-6, IL-8, and TNF-α mRNA levels in AT through lifestyle intervention involving hypocaloric diet and daily moderate activity. These changes were accompanied by a reduction in plasma C-reactive protein, IL-6, IL-8, and monocyte chemoattractant protein-1 (37). With regard to plasma IL-18, it has been shown that lifestyle intervention with diet alone (31) or in combination with physical exercise (32), leading to a moderate weight loss, decreases plasma IL-18 by 25% to 50%. In addition, aerobic exercise has been shown to reduce plasma IL-18, independently of lowering in BMI (38). This suggests that plasma IL-18 is highly sensitive to lifestyle changes (9, 32), but AT IL-18 expression has, to our knowledge, not previously been studied in relation to acute exercise or exercise training. This study showed that 8 weeks of high-intensity training lowered AT IL-18 mRNA expression by 20% in both male and female obese subjects. Interestingly, the reduction in AT IL-18 mRNA expression was not accompanied by improved insulin sensitivity, weight loss, or improvement in blood lipid profile. We speculate that 8 weeks of training simply was not sufficient to obtain such changes and that the reduced AT IL-18 mRNA levels reflect the initiation of an adaptive protection against insulin resistance and systemic inflammation with exercise training, which may contribute to the beneficial effects of regular physical activity on insulin sensitivity.

With regard to AT gene expression associated with acute exercise, limited information is available, although it has been shown that IL-6 mRNA levels and Visfatin mRNA levels increase in abdominal AT after acute exercise in healthy human subjects (39, 40). Interestingly, an acute up-regulation of plasma IL-18 has been shown by hyperglycemia (18), and prolonged (∼10 hours) strenuous endurance exercise reduced plasma IL-18 content 24 hours after exercise in male athletes (41). In this study, however, we did not observe any effect of an acute prolonged exercise bout on AT IL-18 mRNA expression within 10 hours of recovery.

In summary, this study shows that obesity is associated with elevated abdominal AT IL-18 mRNA expression and plasma IL-18 levels and that exercise training decreased AT IL-18 mRNA expression. Because insulin resistance also seemed to be associated with elevated AT IL-18 levels, we suggest that the training-induced reduced IL-18 gene expression in adipose tissue may play a role in improved insulin sensitivity with regular physical activity.

Acknowledgments

We thank the subjects for extraordinary effort and Kristina Møller Kristensen, Hanne Villumsen, Ruth Rovsing, and Carsten Nielsen for skillfull technical assistance. This study was supported by a grant from The Danish Ministry of Culture Committee on Sports Research (Kulturministeriets Udvalg for Idrætsforskning). The Centre of Inflammation and Metabolism is funded by the Danish National Research Foundation (02-512-55). The Copenhagen Muscle Research Centre is supported by grants from The Copenhagen Hospital Corporation, The University of Copenhagen, and The Faculties of Science and of Health Sciences at University of Copenhagen.

Footnotes

  1. Nonstandard abbreviations: IL, interleukin; TNF-α, tumor necrosis factor-α; AT, adipose tissue; WHR, waist-to-hip ratio; HOMA-IR, insulin sensitivity as measured by the homeostasis model assessment; SE, standard error; Vo2max, maximal oxygen uptake; RT, reverse transcription; PCR, polymerase chain reaction; FFA, free fatty acid; TG, triglyceride.

  2. The costs of publication of this article were defrayed, in part, by the payment of page charges. This 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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