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INFLAMMATION/IMMUNITY/MEDIATORS
1Department of Gastroenterology, Hospital Clínic of Barcelona, University of Barcelona; and 2Digestive System Research Unit, University Hospital Vall d'Hebron, Barcelona, Spain
Submitted 20 September 2005 ; accepted in final form 13 July 2006
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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inflammatory bowel diseases; probiotics; cell adhesion molecules; endothelium
Probiotics are defined as viable microorganisms that exhibit a beneficial effect on the health of the host by improving its intestinal microbial balance (17) or, more broadly, as "living micro-organisms, which upon ingestion in certain numbers, exert health effects beyond those inherent to basic nutrition" (14). Various studies have demonstrated the safety and efficacy of probiotic organisms in the treatment or prevention of certain infectious diarrheal diseases (13), in experimental and human IBD (15, 30), in pouchitis (11), in inhibition of gastric colonization by Helicobacter pylori (4), and in lactose intolerance, food allergy, and constipation (19). Promising probiotic strains include the members of genera Lactobacillus, Bifidobacterium, and Enterococcus, some strains of Escherichia coli, and nonbacterial microorganisms such as Saccharomyces boulardii (17). Multiple mechanisms have been suggested to explain the protective effects of probiotics, including inhibition of bacterial growth, reduction of pathogen adhesion, improvement of epithelial barrier integrity, and modulation of immune function (34).
In the present study, we used the probiotic bacteria Lactobacillus casei, which has been proven effective in improving murine chronic IBD and is associated with downregulation of proinflammatory cytokines such as IL-6 and IFN-
in lamina propria mononuclear cells (22). Moreover, L. casei has been demonstrated to be effective preventing the inhibition and invasion of adherent-invasive E. coli to human intestinal epithelial cells isolated from CD patients (16). A previous study showed that colonization of an isolated colonic segment by L. casei attenuated the lesion induced by 2,4,6-trinitrobenzenesulfonic acid (TNBS) in Sprague-Dawley rats and significantly reduced bacterial translocation to the mesenteric lymph nodes, liver, and spleen (21). In the present study, we used L. casei before the induction of colitis by TNBS in rats with a colonic segment surgically excluded from fecal transit. The excluded colonic segment allowed radical elimination of commensal flora and recolonization with preselected bacterial species. Using this in vivo model, we characterized the effect of L. casei administration on the expression of endothelial cell adhesion molecules and leukocyte-endothelial interactions in the colonic microcirculation and evaluated their protective effect.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Surgical procedures and induction of colitis. Rats with a colonic segment excluded from fecal transit were randomly distributed into three groups: control, colitis, and colitis-L. casei (n = 5–7 animals per group and per experiment). An excluded colonic segment was surgically prepared as previously described (21). In ketamine-xylazine-anesthetized rats [90 mg/kg ketamine (Parke-Davis, Ann Arbor, MI) and 10 mg/kg xylazine (Bayer, Leverkusen, Germany)], the abdomen was opened through a midline laparotomy. The terminal ileum at the level of the ileocecal junction and the distal colon was carefully transacted with cecum removal. Previously, colonic vessels were tied with a ligature with Dexon 5-0 (Braun). This part of the colon, i.e., the ascending colon and proximal third of the distal colon, was brought out through two colostomies (i.e., the proximal and distal end) on the left side of the abdominal wall using 4-0 Vicryl suture (Ethicom, 2000 Nordested, FRG). The continuity of the gastrointestinal tract was achieved by an end-to-side ileocolonic anastomosis using 7-0 Vicryl suture (Ethicon) interrupted, single-layer suture. The excluded colon was washed several times using warm (37°C) sterile saline until it became clean. After the abdomen wall was sutured, colostomies were flushed with broad-spectrum antibiotics (50 mg/ml imipenem; Merck, Sharp, & Dohme, Alcalá de Henares, Spain) and 50 mg/ml vancomycin (Dista, Alcobendas, Spain). Rats were then allowed to recover.
One day after surgery, rats were lightly anesthetized with ketamine-xylazine, and the lumen of the excluded segment was thoroughly flushed with a solution of broad-spectrum antibiotics (imipenem and vancomycin) and kept in the lumen for 3 h. Thereafter, the lumen was abundantly rinsed with 60 ml of sterile saline to eliminate the antibiotics. Then, 3 ml of a suspension of aerobic bacteria were inoculated into the segment lumen. The suspension was prepared as described below and included Enterobacter aerogenes, Klebsiella pneumoniae, and Streptococcus viridans for the colitis and control groups and the same strains plus L. casei strain DN-114 001 for the colitis-L. casei group. Colostomies were immediately closed by ligature after inoculation.
On day 3, colostomies were reopened and lumen mucin aggregates were removed by rinsing with 10 ml of Schaedler reduction medium (Bio Mérieux, Marcy l'Etoile, France) to avoid obstruction. Thereafter, 3 ml of anaerobic bacteria suspension were inoculated. The suspension was prepared as described below and included Bacteroides fragilis and Bacteroides disastonis for the colitis and control group and the same strains plus L. casei strain DN-114 001 for the colitis-L. casei group. Colostomies were immediately closed by ligature. To obtain a proper anaerobic viability and colonization, oxygen-intolerant anaerobes had to be inoculated following aerobic inoculation, thus allowing the latter to metabolize the oxygen present in the isolated intestinal loop. The resulting free-oxygen environment was then appropriated for the survival of anaerobic bacteria.
On day 6 after surgery, L. casei colonization in the isolated segment was controlled in all rats by microbiological culture, as described below. Colonization of the nonprobiotic inoculated flora was determined in randomly selected rats, since their ability to colonize the intestinal lumen has already been described in the same experimental model in a previous study (21). The luminal mucus was again removed by rinsing with up to 10 ml of Schaedler broth reduction medium (37°C). One-milliliter aliquots of the effluent were carefully collected in sterile plastic syringes, avoiding contact with air. They were immediately processed for microbiological culture to assess bacterial colonization. The microbiological washing outcomes were semiquantitative as the volume required for flushing removal of mucin aggregates fluctuated. This fluctuation depends on the size of the mucus, consistence, and adherence to the colonic mucosa. The removal of mucus aggregates is of vital importance because they may plug the colonic segment, impeding further liquid flow.
Thereafter, following effluent sampling, colitis was induced in rats from colitis and colitis-L. casei groups by an intraluminal instillation of 3 ml of TNBS solution (45 mg/ml in 10% ethanol) (TNBS from Sigma, St. Louis, MO) and ethanol (from Panreac, Barcelona, Spain).
On day 7 after surgery, leukocyte-endothelial cell interactions were assessed in vivo in colonic venules by intravital microscopy or expression of endothelial ICAM-1 or VCAM-1 was measured by the dual-labeled antibody technique.
Bacterial suspensions. Bacteria used in the present study were kindly provided by the Digestive System Research Unit, University Hospital Vall d'Hebron.
Bacterial suspensions for inoculation were prepared from cultures of each individual strain. Nonprobiotic strains used in this protocol had been previously isolated by microbiological culture from colonic wall homogenates of rats subjected to acute TNBS colitis (8). These isolates include gram-positive and gram-negative aerotolerant and facultative aerobes and gram-negative obligate anaerobes that are commonly found among predominant species of the rat microflora: E. aerogenes, K. pneumoniae, S. viridans, B. fragilis, and B. disastonis (8). Aerotolerant bacteria were grown in liquid culture medium at 37°C under a controlled atmosphere (5% CO2): L. casei and S. viridans were grown in De Man Rogosa Sharp medium (MRS; Difco, Detroit, MI), and E. aerogenes and K. pneumoniae were grown in a 1% tryptone peptone medium (Difco), 0.5% NaCl, and 0.5% yeast extract (Difco). B. fragilis and B. disastonis were grown in Schaedler medium (Difco) for 48 h at 37°C in an anaerobic atmosphere (Anaerocult A Merck, Darmstadt, Germany).
At the end of the phase of exponential growth, bacterial cultures were stopped. The final aerobic inoculum concentration for the three groups of rats were 6 x 109 colony-forming units (CFU)/ml. In the control and colitis groups, 1 ml of a 6 x 109 CFU/ml bacterial culture for each of the three bacteria (E. aerogenes, K. pneumoniae, and S. viridans) were mixed, with a final volume of 3 ml. In the colitis-L. casei group, 0.75 ml of a 6 x 109 CFU/ml bacterial culture for each of the four bacteria (E. aerogenes, K. pneumoniae, and S. viridans plus L. casei) were mixed, again with a final volume of 3 ml. Volumes of 3 ml per rat were instilled into the lumen of the excluded colonic segment on day 1.
The final anaerobic inoculum concentration for the three groups of rats was 5 x 109 CFU/ml. In the control and colitis groups, 1.5 ml of a 5 x 109 CFU/ml bacterial culture for each of the two bacteria (B. fragilis and B. disastonis) were mixed, with a final volume of 3 ml. In the colitis-L. casei group, 1 ml of a 5 x 109 CFU/ml bacterial culture for each of the three bacteria (B. fragilis and B. disastonis, with the L. casei strain) were mixed, again with a final volume of 3 ml. Volumes of 3 ml per rat were instilled into the lumen of the excluded colonic segment on day 3. The concentration of anaerobic was slightly lower than aerobic bacteria because strict anaerobes such as B. fragilis and B. disastonis have a slower growth rate and the final bacteria concentration was also slightly lower in this case. In the present study, probiotic bacteria were not given in higher concentrations, because the aim was to determine whether L. casei was able to compete with other enteric bacteria of the common flora at the same concentration. Llopis and colleagues (21) demonstrated that L. casei is not only able to colonize competing with other common enteric bacteria but is also able to display its beneficial effect in in vivo studies.
Microbiological studies. Samples of washings were collected under sterile conditions before the induction of colitis. Serial decimal dilutions (from log –1 to –8) were performed in sterile saline and inoculated in petri dishes of selective media. L. casei was differentiated in MRS (Difco) at 37°C for 72 h in a microaerobic atmosphere (5% CO2) and selected for colony color and morphology. After incubation, the colonies were counted and further identified by biochemical methods (Gram's staining, catalase, and oxidase) and API 50 CH gallery (Bio Mérieux, l'Etoile, France) for the identification of L. casei.
In vivo assessment of leukocyte-endothelial cell interactions in colonic venules.
Leukocyte-endothelial cell interactions in colonic submucosa and lamina propria venules were characterized by intravital microscopy in the excluded colonic segment. For intravital microscopy studies, rats were anesthetized with a mixture of ketamine (90 mg/kg) and xylazine (10 mg/kg) ip, the right carotid artery was cannulated, and the abdomen was opened via a midline incision. A segment of the excluded colonic loop was chosen for microscopy examination, exteriorized gently, and covered with cotton gauze soaked with bicarbonate buffer. Rats were then placed on an adjustable microscope stage, and the colon was extended over a nonautofluorescent coverslip that allowed observation of a 2-cm2 segment of tissue. Throughout the experiment, the temperature of the animal was maintained with an infrared heat lamp. An inverted microscope (Diaphot 300; Nikon, Tokyo, Japan) with a CF Fluor x40 objective lens (Nikon) was used. A charge-coupled device (CCD) camera (model XC-77 Hamamatsu Photonics; Hamamatsu, Japan) with a C2400 CCD camera control unit and a C2400-68 intensifier head (Hamamatsu Photonics), mounted on the microscope, projected the image onto a monitor (Trinitron KX-14CP1; Sony, Tokyo, Japan) for offline analysis. A video date-time generator (SR-S368E; JVC, Tokyo, Japan) displayed these parameters on recorded and live images. Leukocytes were in vivo labeled by subcutaneous injection of rhodamine 6-G (Molecular Probes, Leiden, The Netherlands) as previously described (36). Rhodamine-6G-associated fluorescence was visualized by epi-illumination at 510–560 nm, by using a 590-nm emission filter. Single unbranched submucosa and lamina propria venules with internal diameters (D) ranging between 25 and 35 µm were selected for observation. The flux of rolling leukocytes, leukocyte rolling velocity, and numbers of adherent leukocytes in 100 µm of venule were determined offline after playback of videotapes. Rolling leukocytes were defined as those white blood cells that moved at a velocity less than that of free-flowing leukocytes (FFV) in the same vessel. The flux of rolling leukocytes was measured as the number of rolling leukocytes that passed a fixed point within a small (10 µm) viewing area of the vessel in a 1-min period. Leukocyte rolling velocity was measured as the time required for a leukocyte to traverse a 50-µm distance along the length of the venule and was calculated as the mean of 10 rolling leukocyte velocities and expressed in micrometers per second. Leukocytes were considered adherent to the venular endothelium when stationary for 30 s or longer and were expressed as number per 100 µm length of venule. Venular blood flow (VBF) was estimated from the mean of the velocity of three FFV using the empirical relationship of VBF = FFV/1.6 (6). Venular wall shear rate () was calculated, assuming cylindrical geometry, using the Newtonian definition of = 8 (VBF/D) (20). In each animal, three to six random venules were examined, and values for number of rolling and adherent leukocytes, leukocyte rolling velocity, and venular wall shear rate were calculated as the mean of each parameter in all venules examined.
Endothelial VCAM-1 and ICAM-1 expression. The monoclonal antibodies (MAb) used to measure VCAM-1 expression were 5F10, a murine IgG2a against rat VCAM-1 (33), and UPC-10, a nonbinding, murine IgG2a. The MAbs used to measure ICAM-1 expression were 1A29, a mouse IgG1 against rat ICAM-1 (41), and P-23, a murine IgG1 directed against human (but not rat) P-selectin. 5F10 was obtained from Biogen (Cambridge, MA) and UPC-10 was from Sigma Química. 1A29 and P-23 were scaled up and purified by protein A or G chromatography at Pharmacia Upjohn Laboratories (Kalamazoo, MI). The binding MAb 5F10 directed against VCAM-1 and 1A29 directed against ICAM-1 were labeled with 125I, whereas the nonbinding MAbs were labeled with 131I (Amersham Ibérica, Madrid, Spain). Radioiodination of the MAbs was performed by using the iodogen method as described previously (7, 27).
Animals were anesthetized with a mixture of ketamine (90 mg/kg) and xylazine (10 mg/kg) ip, and the left jugular vein and the right carotid artery were cannulated with polyethylene-10 tubing (Portex, Hyte, UK).
To measure VCAM-1 expression, 20 µg of 125I-5F10 were administered and 5 µg of 131I-UPC-10 were added to the mixture. For assessment of endothelial ICAM-1, a mixture of 5 µg of 125I-1A29 and 245 µg of unlabeled 1A29 was used, and 5 µg of 131I-P-23 were added to the mixture. The mixture of binding and nonbinding MAbs was administered through the jugular vein catheter. The injected activity in each experiment was calculated by counting a 5-µl sample of the mixture containing the radiolabeled MAbs. After injection, the MAbs were allowed to circulate for 5 min, and the animals were exsanguinated. The accumulated activity of each MAb in an organ was expressed as nanograms of binding antibody per gram of tissue. The formula used to calculate VCAM-1 and ICAM-1 expression was as follows: endothelial expression = [(cpm 125I organ·g–1·cpm 125I injected –1) – (cpm 131I organ·g–1·cpm 131I injected–1) x (cpm 125I in plasma)/(cpm131I in plasma)]·ng injected MAb, where cpm is counts per minute. This formula was modified from the original method (26) to correct the tissue accumulation of nonbinding MAb for the relative plasma levels of binding and nonbinding MAbs (18).
Pathology studies. After rat euthanasia, the isolated segment was extracted and opened longitudinally. A picture was taken of each segment, and a numerical code was assigned. The extent of tissue damage was evaluated by using the image management software Leica IM 50-Measurement (Leica Imaging Systems, Cambridge, UK). The extent of the damaged area was expressed as the percentage of total mucosal surface in the excluded colonic segment. The degree of inflammation of the colon was graded semiquantitatively from 0 to 11, according to previously defined criteria of Appleyard and Wallace (1) and taking into account 1) loss of mucosal architecture (score 0–3); 2) cellular infiltration (score 0–3); 3) muscle thickening (score 0–3); 4) crypt abscess formation (score 0–1); and 5) goblet cell depletion (score 0–1).
Statistical analysis. Data were analyzed by the nonparametric Kruskal-Wallis or Mann-Whitney U-test where appropriate. All values are expressed as means ± SE. Statistical significance was set at P < 0.05.
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RESULTS |
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Nonprobiotic bacteria were also recovered in 100% of the randomly selected rats analyzed, thus indicating that the strategy used for intestinal colonization was highly effective. Pooled bacterial recovery results from the three groups were as follows: Enterobacteriae (K. pneumoniae and E. aerogenes) were recovered from 2 x 102 to 2 x 107 CFU/ml of washing efflux. Streptococcus were recovered from 2 x 102 to 4 x 107 CFU/ml; and Bacteroides (B. disastonis and B. fragilis) were recovered from 1 x 101 to 4 x 107 CFU/ml. Presence of L. casei in the colitis-L. casei group ranged from 4 x 101 to 1.3 x 105 CFU/ml. The wide ranges of these values are most probably related to fluctuations in the flushing volumes used to remove mucin aggregates before sampling. As a consequence, microbiological washing outcomes were semiquantitative.
Leukocyte-endothelial cell interactions in colonic venules. Induction of colitis by administration of TNBS-ethanol in the excluded colonic segment colonized by normal rat colonic flora induced a pronounced increase in leukocyte-endothelial cell interactions (Figs. 1 and 2). There was a onefold increase in the flux of rolling leukocytes (rolling cells per minute) and in the number of rolling leukocytes (numbers of cells rolling in 100-µm length of venule). Few adherent leukocytes were present in venules of noninflamed colonic segments, and an eightfold increase in leukocyte adhesion was observed after administration of TNBS-ethanol in the excluded colonic segment. No differences in leukocyte rolling velocity, venular blood flow, or venular wall shear rate were observed between control and colitis group rats (data not shown).
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Endothelial VCAM-1 and ICAM-1 expression. Endothelial ICAM-1 and VCAM-1 expression significantly increased in the colonic endothelium in association with the induction of colitis. As shown in Fig. 3, under baseline conditions, VCAM-1 expression was low and markedly increased in the inflamed colon (3-fold). Expression of ICAM-1 was significantly higher than that of VCAM-1 under baseline conditions, as estimated by the amount of antibody bound per gram of tissue. The magnitude of ICAM-1 upregulation in colitic animals was weaker than that of VCAM-1.
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To determine whether expression of ICAM-1 and VCAM-1 in the isolated colonic segment might be influenced in part by surgical manipulation, expression of these adhesion molecules was also measured in the proximal ileum and distal end colon. As shown in Table 1, in noncolitic control group animals, no significant differences were observed in the level of expression of ICAM-1 or VCAM-1 between the isolated colonic segment and adjacent segments. Furthermore, induction of colitis by TNBS induced an upregulation of these adhesion molecules only in the isolated loop, indicating that this process does not have a systemic component.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Furthermore, a recent study using the same experimental model and bacterial strains employed in the present study documented a significant reduction in the colonic inflammatory damage induced by TNBS when the excluded segment was inoculated with L. casei and normal flora. Moreover, the addition of the probiotic significantly reduced the translocation of bacteria to the mesenteric lymph nodes, liver, and spleen (21).
In keeping with these observations, we also documented a significant reduction in the severity of gross colonic damage, and this reduction was of similar magnitude to that observed in the previous study. However, colonization by L. casei did not result in significant attenuation of the microscopic lesion. This discrepancy has also been noted in previous studies. For example, Pavan and colleagues (28) found that pretreatment with a L. plantarum or L. lactis strain had no effect on histological scores in mice with TNBS-induced colitis, whereas translocation of bacteria was significantly reduced. This may be related to the fact that histology score is a reflection of the most severe area of damage, and, in our experiments, areas of severe damage were still present in animals administered L. casei, but the area of ulceration was significantly reduced.
The present study was particularly focused on elucidating whether the modulation of the process of leukocyte recruitment was part of the effects of the probiotic treatment. Both experimental (31) and clinical studies (9) have demonstrated that selectively reducing the recruitment of leukocytes into the inflamed intestine affords a significant therapeutic benefit in IBD, and this effect may also be a contributor to the beneficial actions of some drugs in colitis such as cyclosporin A (40).
Recruitment of leukocytes from the vascular compartment to the extravascular space is a critical component of inflammation. Leukocyte trafficking through the vascular wall results from a complex series of interactions between leukocytes and endothelium, which include the following steps: rolling, activation, firm adhesion, and emigration (25).
An enhanced leukocyte recruitment in intestinal venules with significant increases in the numbers of leukocytes with rolling and adhesion interactions has been a constant finding in all experimental models of colitis (12, 31, 39).
These interactions are precisely regulated by several cell adhesion molecules expressed on the surface of endothelial cells and leukocytes (31). Our results show that leukocyte-endothelial cell interactions were attenuated in response to treatment with the probiotic L. casei, with a significant reduction of the number of firmly adherent leukocytes.
Treatment with L. casei did not impact the first phase of leukocyte-endothelial cell interactions, namely leukocyte rolling. Lack of effect of L. casei treatment on rolling interactions probably does not limit the anti-inflammatory action of this treatment, because interfering with this initial cellular interaction in the setting of TNBS-induced colitis does not affect subsequent steps of leukocyte recruitment, such as firm adhesion to the venular endothelium and emigration (32).
Firm adhesion is the crucial determinant of the infiltration of an organ by inflammatory cells. In fact, it has been shown that 80% of cells that adhere for a period of 30 s or longer finally emigrate into the interstitium of an organ (24), and this was the time period used in the present study to categorize a leukocyte as firmly adherent.
The different steps of leukocyte recruitment are governed by adhesion molecules belonging to different families. Thus the initial rolling interactions are mediated by selectins and their ligands, whereas firm adhesion is mediated by integrins on the surface of leukocytes and immunoglobulin superfamily molecules on the endothelium (25). Because intravital microscopy studies revealed that the probiotic L. casei significantly reduces leukocyte adhesion, without having an impact on rolling interactions, to further explore the mechanistic basis for the effects of the probiotic strain on intestinal inflammation, we concentrated our efforts on endothelial cell adhesion molecules involved in firm adhesion.
In that regard, previous studies using the TNBS model of colitis showed that immunoneutralization of ICAM-1 attenuates, but does not completely abrogate, leukocyte adhesion, whereas VCAM-1 blockade decreases leukocyte adhesion in colitic rats to levels similar to those of noncolitic animals (31). Therefore, we studied the effects of L. casei on expression of these two adhesion molecules in colonic inflammation. Interestingly, expression of ICAM-1 was downregulated by treatment with the probiotic, although VCAM-1 expression was unaffected. Therefore, it is conceivable that the reduction of leukocyte adhesion in response to treatment with L. casei is related to the downregulation of ICAM-1. Although this may appear to contradict our previous observations, suggesting a crucial role for VCAM-1 in the pathogenesis of colitis induced by TNBS, it can be explained by an important difference between our present and previous studies in the time point chosen. In the present study we assessed parameters of mucosal inflammation 1 day after TNBS administration. This may represent a more acute phase of the colonic inflammatory reaction, and ICAM-1 is known to play a determining role in acute inflammatory conditions. However, VCAM-1 is more relevant as a mediator of chronic inflammatory diseases in which recruitment of lymphocytes, as opposed to neutrophils, is the main cellular event.
Our study has several limitations. First, in this model, we assessed inflammatory changes within the first 24 h after TNBS administration and, therefore, may not accurately represent the chronic colitis of human IBD. We did not extend this study for a longer period of time because we observed an increment of bacterial contamination together with a reduction in the recovery of the probiotic bacteria. Second, probiotics have been shown to be able to affect various components of the inflammatory cascade: coculture with L. casei strain significantly reduces TNF-
release in normal and inflamed mucosa from patients with CD (3) and also reduces IL-8 release via TNF- independent pathways (2). Therefore, we cannot establish a cause-effect relationship between the downregulation of ICAM-1 and reduction of leukocyte adhesion, because other components of the inflammatory response such as a decrease in leukocyte activating or chemotactic signals may also contribute to reduce leukocyte recruitment in our model.
The use of probiotics in the clinical setting remains controversial, and their mechanism of action has not been fully clarified. We believe our study sheds a beam of light on the mechanistic basis for testing probiotics in a clinical setting. Issues to be resolved include the preparation to use, mode of delivery, and interactions between probiotic bacteria and other traditional medications. Further trials are needed to establish the precise role of probiotic bacteria in the treatment of IBD.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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