• Volume 72 , Number 1
  • Page: 16–26
ORIGINAL ARTICLE

IL-10 treatment of macrophages bolsters intracellular survival of Mycobacterium leprae

Yasuo Fukutomi1; Masanori Matsuoka1; Fumishige Minagawa1; Satoshi Toratani1; Gregory McCormick2; James Krahenbuhl2




ABSTRACT

In these studies, metabolically active Mycobacterium leprae were maintained for as long as 8 weeks in monolayer cultures of mouse peritoneal macrophages (MΦ). Supplemental IL­10, but not TGFβ, bolstered, directly or indirectly, M. leprae metabolism in mouse MΦ. In the cell culture system temperature setting is extremely important and 31 to 33°C incubation temperature was more permissive than 37°C. Acid fast staining and transmission electron microscopy (TEM) of intracellular M. leprae revealed visible elongation of bacilli cultured under the above ideal conditions.



RÉSUMÉ

Mycobacterium leprae n'a jamais encore été vraiment cultivé sur milieu artificiel. Comme M. leprae préfère vivre in vivo à l'intérieur de la cellule, nous avons exploré la croissance in vitro de M. leprae dans des cultures de phagocytes mononucléés ou de macrophages, qui représentent la cellule hôte favorite du bacille de la lèpre. Notre approche ex­périmentale a tenu compte des faiblesses de ce type d'expérimentation : à savoir la viabilité de l'inoculum et la longue durée de multiplication de M. leprae. Le but n'était pas ici de dé­montrer une augmentation mesurable et significative du nombre de M. leprae, mais plutôt le maintien prolongé du métabolisme de M. leprae, en mesurant l'oxydation de l'acide palmi­tique radiomarqué, comme indicateur de viabilité. Des M. leprae ayant un métabolisme actif ont été maintenues jusqu'à 8 semaines dans des cultures monocouches de macrophages péritonéaux de souris. L'ajout d'IL10, mais pas de TGF, a augmenté, directement ou indirectement, le métabolisme de M. leprae dans les macrophages de souris. Le réglage de la température des cultures cellulaires est extrême­ment important et une température d'incubation de 31–33°C était plus favorable qu'une température de 37°C. La coloration acidoalcoolorésistante et la microscopie électronique à transmission des M. leprae a permis de mettre en évidence des élongations bien visibles parmi les bacilles cultivés dans les conditions idéales mentionnées cidessus.



RESUMEN

Mycobacterium leprae todavía no se ha podido cultivar en medios artificiales. Como esta bacteria prefiere una existencia intracelular in vivo, en este estudio exploramos el crec­imiento in vitro de M. leprae en cultivos de fagocitos o macrófagos, el huésped preferido del bacilo de la lepra. Reconocemos que nuestro diseño experimental conlleva dos problemas: la viabilidad de M. leprae en el inóculo, usualmente baja, y el prolongado tiempo de división de la bacteria. Aunque no esperábamos encontrar un incremento sustancial en los números de M. leprae, sí pensamos poder observar cambios en su metabolismo midiendo la oxi­dación del ácido palmítico radiactivo como un marcador de viabilidad. Encontramos que la bacteria se mantuvo metabolicamente activa hasta por 8 semanas en los cultivos de los macrófagos peritoneales de ratón. La adición de IL10 pero no de TGF, apoyó, directa o in­directamente el metabolismo de M. leprae en los macrófagos de ratón. Las condiciones de incubación de los cultivos fueron muy importantes y la temperatura de 31–33°C fue más permisiva que la temperatura de 37°C. La tinción para ácidoresistentes y la microscopía electrónica de transmisión revelaron cierto grado de alargamiento de los bacilos bajo las condiciones óptimas de cultivo de los macrófagos.





In the 130 years since the discovery of Mycobacterium leprae as the causative agent of leprosy, a large number of attempts have been made to cultivate this obligate intracellular pathogen in cellfree media (6, 30). None of these efforts have fulfilled the criteria for success suggested by John Hanks (18), especially the confirmation of findings by a second laboratory. The inability to culture the leprosy bacillus has undoubtedly hindered almost every aspect of leprosy research, and thus, our understanding of this disease lags behind that of many others of bacterial etiology.

Reasoning that the intracellular milieu would best suit the multiplication of the obligate intracellular leprosy bacillus, as an alternative to culture in axenic medium, a number of attempts have been made to cultivate M. leprae in various types of cultured cells. Fieldsteel and McIntosh (10) employed a range of rat, mouse, and human tissue but found no evidence of multiplication. The preferred host cell for the leprosy bacillus, however, appears to be the mononuclear phagocyte or macrophage (MФ) and a number of unsuccessful attempts have been made to grow M. leprae in MФ (5, 8, 16, 24, 27, 31, 34, 36, 41). The present approach also employs MФ but is novel in that we employed conditions that inhibit innate antimicrobial functions in infected mouse MФs to bolster the intracellular survival of M. leprae. Moreover, we had a number of advantages over the previous attempts by others including: unique resources, previously unavailable to other workers in the form of fresh, highly viable M. leprae (42), sensitive techniques for measuring and comparing the metabolic activity of M. leprae (13) and the extensive experience of our two laboratories in studying the relationship between the MФ and the leprosy bacillus (2, 14, 15, 39, 40).

 

MATERIALS AND METHODS

Maintenance of a viable M. leprae inoculum. The Thai53 strain of M. leprae (26) was maintained in continuous passage in athymic nu/nu mice (Crea Co., Tokyo, Japan) by inoculation of 1 x 107 freshly harvested bacilli into both hind footpads. At approximately nine months post, footpads were processed to recover M. leprae by Nakamura's method with a slight modification (28). Briefly, tissue was minced and homogenized with Hanks' balanced salt solution (HBSS) containing 0.05% Tween 80. The homogenate was centrifuged at 150 x g for 10 min and supernatant of the sample homogenate was treated with 0.05% trypsin at 37ºC for 60 min. The suspension was centrifuged at 4000 x g for 20 min and sediment was resuspended in HBSS followed by treatment with 1% sodium hydroxide at 37ºC for 15 min. The treated material was washed and resuspended in HBSS at the desired bacillary concentration. Bacillary number in each footpad was enumerated individually according to standard techniques (37).

Cytokines. Murine recombinant IL10 was obtained from Genzyme Corp. T cell growth factor β (TGFβ) was obtained from Kurashiki Bouseki, Kurashiki, Japan). Both cytokines were stored at -80ºC until use.

Mouse MФ culture. Mouse peritoneal resident cells (approximately 50% MФ) were harvested from retired ICR or Swiss White (SW) mice and suspended as previously described (2) at a concentration of 2 x 106/ml in RPMI 1640 (GIBCO, Grand Island, NY) + 15% fetal bovine serum (HyClone Laboratories, Logan, UT) + 25 mM hydroxyethylpiperazineN'2ethanesulfonic acid (HEPES) (GIBCO), 0.2% NaHCO3 (GIBCO), 2 mM glutamine (Irvine Scientific, Santa Ana, CA), and 100 μg/ml ampicillin (Sigma Chemical Co., St. Louis, MO). 0.5 ml was seeded into 24 well tissue culture plates (Corning) containing 16 mm LUX coverslips (Miles Laboratory, Napierville, IL). After overnight adherence of the cells, MФ monolayers were obtained after washing nonadherant cells from the coverslip with Hanks Balanced Salt Solution (HBSS) leaving approximately 1 x 106 MФ adhered per coverslip.

Infection of MФ with M. leprae. Purified mouse MФ monolayers were infected with fresh M. leprae suspended in 0.5 ml medium at a multiplicity of infection of 20:1. After 4 hr incubation, nonphagocytized bacteria were removed by washing and the cultures reincubated in 1.0 ml media supplemented with the appropriate cytokine in 5% CO2 at the appropriate experimental temperatures (2). Media was changed and, where appropriate, cytokines replenished at 5 day intervals.

Radiorespirometry (RR). The MФ were lysed with 0.1 N NaOH to release the M. leprae, and the viability of the bacilli was determined by evaluating the oxidation of 14Cpalmitic acid to 14CO2 by radiorespirometry as described previously (13). Total isotope release was usually analyzed after one week of incubation at 31ºC (2).

Staining of M. lepraeinfected MФ. Coverslips of M. lepraeinfected adherent MФs were prefixed with absolute methanol, and acidfast stained Photomicrographs were taken using a Nikon Optiphot microscope using an oil immersion Plan APO 100 lens.

Transmission electron microscopy (TEM). MФ monolayers on coverslips were prefixed in 2% glutaraldehyde/0.1 M Nacacodylate buffer followed by postfixation with osmium tetroxide/KCN and endoblock staining with uranyl acetate. The specimen was then dehydrated with ethanol, embedded in EponAraldite, sectioned at 90 nm, stained with uranyl acetatelead citrate and viewed with a Philips 410 TEM as previously described (39).

 

RESULTS

In vitro temperature preferences of M.leprae. M. leprae clearly prefers cooler incubation temperatures. As shown in Figure 1A, in axenic culture, it was apparent that 37ºC was not an ideal temperature to demonstrate sustained viability. Incubation at 35ºC was more supportive than 37ºC, and results at 29ºC and 32ºC were indistinguishable but even more ideal.

 

Fig. 1A. Effect of incubation temperature on metabolic activity (RR) of M. leprae in axenic media. Each vial was inoculated in triplicate with 2 x 107 freshly harvested, nude mousederived M. leprae and incubated at different temperatures. In the experiment shown, metabolic activity, shown as 14CO2 release, was monitored every day for 8 days. These findings are representative of dozens of related experiments showing the detrimental effects of 37ºC for M. leprae.

 

Similarly, intracellular M. leprae thrives better at cooler temperatures. Mouse MФ appeared to function normally at 33ºC and even 31ºC, as judged by attachment to plastic and phagocytic capacity, although they did not spread as well at these lower temperatures as they do at 37ºC. In the experiment depicted in Figure 1B, infected MФs were incubated at either 31ºC or 37ºC and at 5 day intervals released bacilli were studied by RR for an additional 7 days. The detrimental effects of incubation at 37ºC on M. leprae metabolism were apparent by day 5. In marked contrast, M. leprae cultured in MФ at 31ºC thrived for at least 15 days and retained most of its viability after 25 days in MФ maintained at the lower temperature.

 

Fig. 1B. Metabolic activity of M. leprae in MФ cultured at 31ºC or 37ºC. M. leprae were released from infected MФ on the days shown (in quadruplicate) and inoculated into RR vials. The data shown represent RR data obtained after 7 days.

 

Effects of cytokines on viability of M. leprae in MФ. Supplementation of the infected MФ culture medium with 2 U/ml murine IL10 was clearly associated with sustained viability of intracellular M. leprae. In the more prolonged experiments depicted in Figure 2A and 2B, M. leprae steadily lost viability in control MФ at 31ºC and 37ºC. In contrast, in MФ incubated in the presence of IL10, M. leprae maintained their viability, but only at the permissive temperature of 31ºC. (Figure 2A and 2B). As shown in Figure 2A, addition of TGFβ to the infected MФ had no effect on the viability of M. leprae.

 

Fig. 2A. Metabolic activity of M. leprae in MФ cultured in the presence of IL10 or TGFβ. M. leprae infected MФ were incubated at 31ºC in the presence or absence of 2 U/ml IL10 or 10 ng/ml TGFβ and bacilli were released from infected MФ on the days shown (in triplicate) and inoculated into RR vials. The data shown represent RR data obtained after 7 days.

 

Fig. 2B. Comparison of metabolic activity of M. leprae in MФ cultured at 31ºC and 37ºC in the presence of IL10. M. leprae infected MФ were incubated at 31ºC or 37ºC in the presence or absence of 2 U/ml IL10 and bacilli were released from infected MФ on the days shown (in triplicate) and inoculated into RR vials. The data shown represent RR data obtained after 7 days.

 

Experiments were run to account for all of the M. leprae in the long term cultures, assuming that during prolonged culture some infected MФ may detach or lyse, releasing their bacilli. In the experiment depicted in Fig. 3, media was changed as usual every 5 days and data points recorded every 10 days. In order to account for bacilli released from MФ or bacilli in "detached" MФ we collected and saved the "old" media at 4ºC at the time it was changed (midpoints of the 10 day time points plotted at 20, 30, 40 days, etc.). The viability of bacilli in the individual MФ monolayers and in the MФ detached from the monolayers are shown separately and as a total. These data show that only a few M. leprae were released or infected cells detached into the supernatant media, and the cumulative radiorespirometry (RR) results from individual wells confirmed the ability of IL10 treatment to sustain intracellular viability of M. leprae.

 

Fig.3. Metabolic activity of M. leprae in MФ cultured in the presence of IL10. M. leprae infected MФ were incubated at 31ºC in the presence or absence of 2 U/ml IL10. On the days shown (starting on day 20) we accounted for the metabolic activity of all bacilli in each well (free bacilli or detached infected cells in supernatant plus bacilli present in attached MФ). The middle open portion of each bar represents RR activity of bacilli released from adherent MФ. The upper shaded portion of each stacked bar represents RR activity of bacilli found in the supernatant when the media was changed 5 days previously (day 15, 25, 35, etc.). The lower black portion of each bar represent RR of bacilli in the supernatant on the day of the harvest.

 

Morphological evaluation of M. leprae with sustained metabolic activity in MФ. The morphological characteristics of M. leprae maintained in prolonged culture in mouse peritoneal MФ were observed with light and electron microscopy. Elongated M. leprae were only observed under conditions where infected MФ were maintained at 31ºC in the presence of IL10. As shown in Fig. 4, acid fast staining of infected MФ at 4 weeks revealed that at 31ºC in the presence of IL10, many of the intracellular M. leprae were clearly elongated in comparison to those seen at 0 time or in MФ maintained at 31ºC without IL10 (Figure 4A, B, C). At 37ºC elongation of bacilli was not observed regardless of the presence of IL10 (Fig. 4D). Under the transmission electron microscope (TEM), elongation was even more apparent. Not all bacilli in the 31ºC, IL10 group were observed to be elongated, as this required all bacilli to be sectioned through their long axis; but examination of dozens of infected cells in 2 experiments revealed elongated cells (8 to 10 μ) only in the 31ºC, IL10 group. M. leprae in the control group were consistently 2 to 4μ in length (Fig. 5).

 

Fig. 4. Elongation of M. leprae in mouse MФ cultured in the presence of IL10 at 31ºC or 37ºC. The cells were acid fast stained and observed under light microscopy with magnification at x 1000. Panel A = 0 hr (31ºC). B = 4 wks at 31ºC without IL10. C = 4 wks at 31ºC + IL10. D = 4 wks at 37ºC + IL10. Figures are representative of observations from 2 experiments.

 

Fig. 5. Elongation of M. leprae in macrophages cultured in vitro. Mouse macrophages were infected with M. leprae followed by washing and incubation in the presence or absence of IL10 at 31ºC for 4 weeks. The cells were fixed, cut and observed under transmission electron microscopy. A, control (without IL10); B, C, +IL10. Bars in each panel = 2 μm.

 

DISCUSSION

Our goals in this study were limited. Convincing evidence of actual intracellular multiplication of M. leprae would require at least a 10fold increase in bacillary numbers. With a calculated multiplication cycle of 12.8 days in the mouse footpad model (37), this minimally acceptable increase in numbers would be difficult to demonstrate in a few weeks of MФ tissue culture. However, the present study did show that the metabolism, and presumably the viability (42), of M. leprae could be sustained under culture conditions which also appeared to support the intracellular elongatiion of the leprosy bacillus.

In vivo M. leprae is able to enter and survive in a wide variety of tissues and cell types (24). Attempts to culture M. leprae in tissue culture have included the use of numerous cell lines derived from humans, rats, and mouse tissue with no evidence of multiplication (10, 27). The MФ, the preferred host cell for the leprosy bacillus, offers an advantage over tissue culture cell lines since MФ actively phagocytize M. leprae and, unlike cell lines, MФ in culture are nondividing adherent cells. Consequently the intracellular status of M. leprae over time is not confounded by an increase in host cell numbers. Chang and Neikirk (5) demonstrated the long term infection of mouse MФ cultures with M. leprae, and a report of success in culturing M. leprae in MФ was made by Garbutt (16), but was not confirmed by McRae and Shepard (27). Others reported limited, questionable, and unconfirmed success at detecting multiplication of the organism in MФ cultures (8, 31, 34, 41). An exhaustive but unsuccessful attempt to cultivate M. leprae in tissue culture was made by Sharp and Banerjee (36) who employed MФ from conventional mice and rats, nu/nu mice and rats and armadillos, rather than dividing cells and cell lines. Their M. leprae inocula was derived from 3 sources (human leproma, nu/nu mouse footpad and frozen infected armadillo tissue). Incubation temperature was varied from 31ºC to 35ºC and infected cells were maintained for up to 200 days. They rigorously evaluated any increase in leprosy bacilli and concluded that no significant multiplication occurred.

Our studies provide groundwork for future rational attempts to cultivate M. leprae with notable advantages to our approach. Most importantly, our starting inoculum of M. leprae was freshly obtained for each experiment from infected nu/nu mice maintained under conditions designed to maximize M. leprae viability (42). We also were able to rapidly quantify the metabolic activity of M. leprae using the RR technique adapted by Franzblau (13). This assay can readily detect activity from as few as 106 bacilli with the results available in 1 wk (compared to 6 to 12 months when titrated in mouse footpads). RR data correlates well with other in vitro systems (13) but, more importantly, as shown in a recent series of 36 separate experiments, RR metabolic data correlated well with "viability" studied in the socalled "gold standard" mouse footpad system (42).

Whether intracellular or extracellular, M. leprae clearly prefers temperatures cooler than normal human body temperature, lending experimental credence to the clinical observations of generations of leprologists regarding the distribution of M. leprae in the cooler, permissive sites in human disease, the skin and mucous membranes of the upper respiratory tract. These studies also confirm and extend the results of dozens of other in vitro experiments (42) where 37ºC appeared to be highly detrimental to M. leprae viability.

Under conditions of an effective celluar immune (CMI) response, MФ are likely a major antimicrobial effector cell in host capacity to cope with the leprosy bacillus. We have previously shown that mouse MФ, activated by interferon (IFNγ), kill or inhibit a wide variety of intracellular pathogens (38), including M. leprae (32). Enhanced fusion of secondary lysosmes with M. lepraecontaining phagosomes occurs in activated MФ (39) and is accompanied by the enhanced production of reactive oxygen (ROI) and nitrogen intermediates (RNI) as potent antimicrobial mechanisms (2). We have subsequently shown in vivo in transgenic knockout mice and in vitro in MФ from these mice that ROI are relatively ineffective in comparison to the potent effects of RNI in host defense against M. leprae (1, 3). However, although the enhanced production of RNI by activated MФ is likely a principal antimicrobial mechanism, in each of our studies, normal MФ produced a measurable baseline level of RNI, insufficient to rapidly kill bacilli but perhaps sufficient to inhibit the long term viability of the fastidious, intracellular leprosy bacillus.

In choosing TGF and IL10 as the cytokines that might bolster the intracellular survival of M. leprae, we were attempting to down regulate any innate ability of the normal MФ to cope with the organism. TGFβ is produced by activated MФ and other inflammatory cells and has a broad array of modulatory functions on the immune response. TGFβ has been shown to interfere with MФ antimicrobial mechanisms including generation of ROI (43) and RNI (7), and has been shown to enhance the intracellular growth of M. tuberculosis in human monocytes (21). However, as employed in the present studies with mouse MФ exogenous TGFβ had no detectable effect on sustaining intracellular M. leprae viability, a finding perhaps attributable to the enhanced innate antimicrobial ability of human monocytes in comparison to human monocyte derived MФ (44) which are more akin to resident mouse peritoneal MФ.

In contrast, supplementing media with IL10 clearly affected the long term viability of M. leprae in mouse MФ. IL10 is produced in TH1 responses by T cells, B cells and MФ (11, 29). IL10 has been shown to be a potent downregulator of CMI to intracellular pathogens (33). In vivo, endogenous IL10 dampened the CMI response to avirulent mycobacterial infection (35) and appeared to lead to loss of control of M. tuberculosis infection with widespread dissemination (9). IL10 functions in part at the level of the macrophage by attenuating inducible nitric oxide synthase (iNOS) mRNA expression, iNOS activity and, by inference, NO production (22). In vitro, exogenous IL10 inhibited production of nitric oxide (NO) in MФ infected with Babesia merozoites (17). In our own studies, IL10 did markedly inhibit the production of NO by IFNactivated MФs but any inhibition by IL10 of baseline production of NO by normal MФs was below the limits of detection of the NO2 assay (data not shown). Exogenous IL10 also interferes with IFNinduced antimycobacterial MФ activities as shown in studies with M. bovis (12).

In addition to sustained metabolism of M. leprae, we also explored a morphological parameter of M. leprae vitality within cultured macrohages, bacillary elongation. Nakamura, et al. reported the elongation of M. lepraemurium in culture medium (28), and in 1969 Chang and Anderson (4) evaluated intracellular growth of M. leprae-murium in cultured mouse peritoneal MФ over a 12 to 17 week period and observed marked elongation of bacilli well before the appearance of bacillary multiplication. In our studies, in addition to sustained or enhanced metabolism, intracellular elongation of individual bacilli was observed after 4 weeks culture in murine MФ maintained with IL10. Elongation was first observed with the light microscope and subsequently confirmed with the transmission electron microscope. A drawback to the use of the TEM for such observations is that unlike light microscopy which revealed the bacilli at their full intracellular length, processing for the TEM required sectioning infected MФ with no certainty that the full length of each bacillus was cut. Nevertheless sufficient numbers of clearly elongated bacilli were seen to confirm the light microscopy findings. Similarly, in preliminary studies with armadillo peripheral blood monocyte derived MФ maintained at 33ºC for 4 weeks, intracellular M. leprae were predominently elongated (data not shown).

Septal formation has been described in TEM studies from human leprosy biopsies by Hirata (20) and was occasionally observed by us in individual bacilli under the TEM after 4 weeks of incubation of infected mouse MФ in the presence of IL10. The small sample size precluded quantification but septa appeared to be observed far less frequently in bacilli in the control MФ. Septal formation in M. leprae murium in the mouse model has been reported to indicate dividing stage of the bacillus, and Hart and Rees (19) concluded that elongation in vitro was an inherent feature of M. leprae murium that distinguishes it from M. leprae although it is likely that the M. leprae inocula employed were of very low viability. Experiments are currently underway to study M. leprae in our cell culture system employing an environmental scanning electron microscope which by passes the need for critical point drying and its attendant artifacts and will permit quantitation of elongation.

Further work with infected armadillo MФ is also clearly warranted. Other than having a core temperature of ~33ºC, little is known about the unique characteristics of Dasypus novemcinctus, the ninebanded armadillo, that render it as a permissive host for the leprosy bacillus (25). In vivo, mononuclear phagocytes in virtually every organ of the natural or experimentally infected armadillo become heavily parasitized with propagating M. leprae (24).

The inability to culture M. leprae has undoubtedly hindered almost every aspect of leprosy research, and thus, our understanding of this disease lags behind that of many others of bacterial etiology. These promising results represent only preliminary findings, but suggest that this approach of inhibiting the innate antimicrobial properties of the MФ to bolster the intracellular survival of M. leprae may ultimately provide clues allowing the long soughtafter cultivation of the leprosy bacillus. In vitro cultivation of M. leprae could make available for the first time, large quantities of pure bacilli produced inexpensively under defined conditions. Thus, large amounts of purified antigens would be available for basic and applied immunological studies, including the development of specific skin test antigens and vaccine preparations. The time and cost of screening new drugs and susceptibility testing of clinical isolates would also be greatly reduced. Our understanding of leprosy epidemiology might increase by determining the existence of human carriers, nonhuman reservoirs, or environmental sources of M. leprae. Phenotypic variation among cultured worldwide isolates could become feasible as might the generation and characterization of mutants. Finally, cultivation of M. leprae, in concert with the genome project, would clearly enhance our understanding of the physiology of this fastidious pathogen, including elucidation of metabolic pathways, studies of virulence mechanisms, drug resistance and the factors underlying "persistence."

Acknowledgment. A Part of this work was financially supported (in Japan) by the Budget for Nuclear Research of the Ministry of Education, Culture, Sports, Science and Technology, based on the screening and counseling by the Atomic Energy Commission and in the U.S.A. by the American Leprosy Missions.

 

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1. Leprosy Research Center, National institute of Infectious Diseases, Tokyo, Japan.
2. The Gillis W. Long Hansen's Disease Center Laboratory Research Branch, Louisiana State University, Baton Rouge, Louisiana, U.S.A.

Reprint requests to
Dr. Y. Fukutomi
Email: fukutomi@nih.go.jp

Received for publication 23 March 2003.
Accepted for publication 30 December 2003.

The JOURNAL would like to thank Ian Orme who served as guest editor for this article.

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