IN-VITRO MODIFICATION O F MEMBRANE RECEPTORS O N CELLS O F T H E MONONUCLEAR PHAGOCYTE SYSTEM MARIANRIDLEY,J. L. TURKAND P. BADENOCH-JONES Department of Pathology, Royal College of Surgeons of England, Lincoln's Inn Fields, London, WC2A 3PN

PLATELXIII RECEPTORS for IgG, complement, phagocytic particles, lectins and macrophage migration inhibition factor (MIF) all represent potential markers for the study of the distribution and mobility of macrophage surface components (Berken and Benacerraf, 1966; Lay and Nussenweig, 1968; Rabinovitch, 1969; Leu et al., 1972; Steinman and Cohn, 1972; Remold, 1973; Oliver, Zurien and Berlin, 1975). Previously, cryostat sections layered with sheep red blood cells (SRBC) for spontaneous E adherence, SRBC+antibody (EA) for Fc receptors and SRBC+antibody+complement (EAC) for C3 showed two patterns in leprosy(Ridley, Ridley and Turk, 1978). EAC but not EA adherence was seen at the BT end of the leprosy spectrum, being particularly dense around epithelioid cells. EA adherence was seen in the active macrophages of BL or LL leprosy. In LL leprosy, EAC adherence was poor or absent. This phenomenon seemed to be related to bacterial density. As it was difficult to interpret the findings on account of the limitations of the section preparations, it was thought that in-vitro studies might help to explain the results and lead to a further understanding of receptor sites in cells of the mononuclear-phagocyte series. This study of macrophage surface receptors, and to a lesser extent those of fibroblasts, following phagocytosis of latex, zymosan, BCG, aluminium hydroxide and treatment with lymphokines, attempts to relate this to certain situations in the leprosy spectrum. MATERIALS AND METHODS A4aterials. Polyvinyltoluene latex particles (2.03 prn diameter, Coulter Electronics Ltd, Harpenden, Herts., UK) were sedimented by cetrifugation, washed in saline and sterilised in 70 per cent. ethanol for 1 hr. A stock suspension was made up in saline and an aliquot added to the cell cultures to a final concentration of 1 mg/ml. Zyrnosan (Sigma London Chemical Co., Poole, Dorset, UK) was added to cultures at a final concentration of 1 mg/ml. BCG vaccine (Glaxo Laboratories, Greenford, Middx., UK) was added to cultures to a final concentration of 5 mg/ml or 10 mg/ml. Aluminium hydroxide (Hopkins &Williams, Romford, Essex, UK) was prepared as described previously (Badenoch-Jones, Turk and Parker, 1978). The amorphous gel was added to cultures at a final concentration of 50 pglml. Lymphokine samples were obtained from Dr R. A. Wolstencroft, Kennedy Institute of Rheumatology, London, W6) as a freeze-dried, ammonium sulphate precipitated material (Dumonde et al., ~~

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Received 25 July 1978; accepted 5 Aug. 1978. J. PATH.-VOL.

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1972). This was added to cell cultures to a final concentration of 62 p.g/ml at the beginning of the culture period. This concentration of lymphokine gave a 50 per cent. inhibition of macrophage migration, in our laboratory. Rabbit anti-sheep whole serum antibody was obtained from the Wellcome Laboratories, Beckenham, Kent, UK. Paraffin oil was obtained from British Drug House, Poole, Dorset, UM. Sheep red blood cells (SRBC) were obtained in Alsevers solution from Wellcome Research Laboratories. Rabbit red blood cells were freshly obtained from rabbits in this department. Cell cultures. Experiments were carried out on non-elicited guinea-pig peritoneal macrophages cultuied for 2 hr, 24 hr and 7 days. For comparison, paraffin oil-induced peritoneal macrophages were also used. Non-elicited macrophages were collected by washing out the peritoneum with 100 ml of medium 199 at 4°C. After washing the cells in this medium they were suspended at 1.5-2.0 x 106 total cells per ml and adhered for 2 hr in plastic Falcon flasks (25 cm2). Nonadherent cells were removed by washing and the adherent cells were cultured in medium 199 supplemented with 50 per cent. normal guinea-pig serum. Oil-induced macrophages were prepared in the same way from guinea-pigs previously injected (3-4 days) i.p. with 20 ml sterile paraffin oil. Adherent cells were recovered, for measurement of EA and EAC rosetting, by gently scraping the cell layer. Guinea-pig skin fibroblasts were obtained from skin explants, as previously described (Badenoch-Jones, Turk and Parker, 1978). They were suspended directly from the flask, by gentle scraping. They were not subcultured. Non-elicited macrophage cultures were sometimes contaminated with fibroblasts, which could be easily recognised by their large size, both as an adherent cell population and in suspension after gentle scraping. Treatment of cell cultures. Measured amounts of latex, zymosan, BCG, aluminium hydroxide and lymphokine were added to macrophages at the start of the culture period. At 2 hr, 24 hr and 7 days non-adherent cells were washed off and discarded, the adherent cells were scraped off the flask and rosetting was carried out on the suspensions. Cell membrane permeability of adherent cells was measured with 0.2 per cent. Trypan blue and by fluorescence with 2 per cent. ethidium bromide and 0-02 per cent. acridine orange. Rosetting. Spontaneous E rosettes using 1.5 per cent. rabbit red cells (Jondal, Holm and Wigzell, 1972) produced no binding to the macrophages. Control experiments for nonspecific binding of sheep red blood cells (SRBC) to macrophages was also negative. Hanks buffered salt solution (HBSS), pH 7.4 was used throughout. For EA rosettes, a suspension of SRBC (1-5 per cent) was incubated with a 1/1500 dilution of rabbit anti-sheep antiserum at room temperature for 30 min. For EAC rosettes, 2 ml of EA was mixed with 0.3 ml of a 1/10 dilution of freshly prepared mouse complement (whole serum) and incubated at 37°C for 30 min. Rosetting was carried out by mixing 0.2 ml of macrophage suspension containing 2 X 106 cells/ml and 0.2ml of EA or EAC suspension for 45 min. at room temperature, after which the suspension was centrifuged for 3 min. at 2009. After resuspension, cytocentrifuge preparations were made and stained with Leishman’s stain. The percentage of cells rosetting was measured by counting at least 200 cells. Some cells were cultured on Leighton tube coverslips, in which case the coverslips were washed three times with HBSS, overlaid with EA or EAC and allowed to rosette at room temperature for 45 min. in a moist chamber. They were rinsed in HBSS to remove unattached cells and stained with Leishman’s stain. Adherent cells were stained with a mixture of ethidium bromide (2 per cent.) and acridine orange (0.02 per cent.) in phosphate-buffered saline and cells identified as macrophages on the basis of staining. Rosette formation was measured as the percentage of adherent cells, identified as macrophages, which formed rosettes.

RESULTS 1. Cell cultures Initially the non-elicited adherent cells were heterogeneous. Staining with Leishman’s stain showed that about 10-20 per cent. were eosinophils and the

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remainder were small cells, spread out and with cytoplasmic processes. After culturing in 50 per cent. serum many of the small cells dropped off and the remaining adherent ceIIs increased in size and became very granular. By day 7 the adherent cells had the appearance of mature macrophages with many processes (fig. 1A and B). These cells were cultured for up to 16 days. By day 12 the cells appeared to reach their maximum maturity with extremely long processes; they rounded up and died by day 16.

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decrease in the percentage of non-elicited adherent macrophages showing EA (@) and EAC (0) receptors after culture of the cells with AI(OH), at 50 pg/ml in the presence of 50 per cent. guinea-pig serum.

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The adherent oil-induced cell preparation was almost entirely macrophages. During culture there was a loss of adherent cells; the appearance of the remaining adherent cells did not change during the culture period. Non-elicited cells cultured in the presence of 20 per cent. or 50 per cent. serum survived better at 7 days than those cultures in 5 per cent. serum. Inactivation of serum (56OCx30 min) or the addition of fresh complement (Wellcome Research Laboratories, Beckenham, Kent) at a dilution of 1/20 or 1/40 at the start of the culture period did not alter cell survival or morphology. On addition of lymphokine at the start of the culture period, there was n o perceptible difference in morphology from the controls at 2 hr, 24 hr or 7 days.

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However, non-elicited macrophages cultured for 7 days and then presented with lymphokine were more basophilic. 2. Phagocytosis

Both non-elicited and paraffin-induced adherent cells readily ingested latex and zymosan. After ingestion there was a decrease in cell volume and spreading. No morphological changes were seen at 24 hr and 7 days after addition of these particles. Cells saturated with latex failed to take up BCG

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FIG.3.-The change in the percentage of non-elicited adherent macrophages showing EA and EAC (0) receptors in the absence (-) or presence (- - - -) of BCG a t 5 mg/ml.

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but the reverse was possible. At the dose used, a third of all non-elicited adherent macrophages and 70 per cent. of paraffin-induced adherent macrophages ingested BCG, as identified by acid-fast staining (Ziehl-Neelsen). At 24 hr after ingestion of BCG the cells showed a ruffled membrane with microspires, many were more spread out, amoeboid and basophilic. The nucleus was central and bacilli were almost always ingested by the polar ends of the cells. About 80 per cent. of non-elicited adherent macrophages showed evidence of ingestion of aluminium hydroxide, as judged morphologically. This caused

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withdrawal of cell processes and rounding up of the cell; the cytoplasm was notably vacuolated. 3. Rosetting No change in the percentage of adherent macrophages rosetting with EA or EAC was noted as a result of altering the serum or complement concentrations of the culture medium. There was no change in EA rosetting, over 7 days of culture, by macrophages after ingestion of latex, zymosan, BCG or treatment

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FIG.4.-The change in the percentage of paraffin-induced adherent macrophages showing EAC receptors in the absence (0-0) or in the presence of 5 mg/ml BCG (0-0) or 10 mg/ml BCG (0-

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with lymphokine. After treatment of cells with AI(OH)3 there was a decrease in both EA and EAC macrophage receptors at 2 hr, 24 hr and 7 days (fig. 2). On the other hand, after treatment with BCG the number of macrophages rosetting with EAC decreased at 24 hr but increased back to 100 per cent. by 7 days (fig. 3). This phenomenon was best seen in macrophages taken from older animals (500 g). With macrophages taken from younger animals (350 g) the number of macrophages showing EAC receptors was decreased by 2 hr after addittion of BCG and this recovered back to 100 per cent. by day 7, This decrease in the number of EAC rosetting macrophages after the addition

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of BCG was also seen with paraffin oil-induced macrophages (fig. 4). In addition non-elicited cells cultured for 7 days and then treated with lymphokine and exposed to BCG showed a similar decrease in EAC but not EA receptors (fig. 5). It is known that there is a relationship between rosetting of SRBC via Fc and C3 receptors and immunological phagocytosis of SRBC carrying these groups (Mariano, Nikitin and Malucelli, 1976).

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DAYS FIG. 5.-The change in the percentage of non-elicited adherent macrophages showing EA (as-.) and EAC (0-0) receptors after culture for 7 days and then the addition of lymphokine and BCG (5 mgiml).

Ingestion of SRBC was thus also measured in addition to rosette formation. It was found that non-elicited macrophages did not ingest EAC-SRBC whether treated or not treated with latex, zymosan or AI(OH)3. However, ingestion of EAC-SRBC occurred after treatment with BCG. In the case of paraffininduced macrophages, both untreated cells and cells treated with BCG showed ingestion of EAC-SRBC. However, paraffin-induced macrophages treated with latex, zymosan and Al(OH)3 showed no ingestion. Ingestion of EASRBC did not occur consistently although there was some ingestion by untreated non-elicited and paraffin-induced macrophages. These results are summarised in the table.

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Experiments were also carried out on the total cell population, as shown in fig. 6. In a cytocentrifuge preparation of the original non-elicited total cell TABLE Ingestion of EAC-SRBC by adherent macrophages after 7 days of culture Additions to cells Control (no addition) BCG Latex Zymosan NOH), Lymphokine

Non-elicited adherent macrophages

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2 hr 2.4 hr 7dY FIG.6.-The change in the percentage of total non-elicited cells showing EA ( 8 )and EAC (0) receptors in the presence (- - -) or absence (-) of BCG at 5 mg/ml. The result at time zero was obtained from cytocentrifuge preparations of the total cell suspension. The results for 2 hr, 24 hr and 7 days refer to total adherent cells.

population, approximately 10 per cent. of cells were identified as macrophages and these all rosetted with both EA and EAC. The original cell suspension was adhered, and non-adherent cells discarded. After culture for 2 hr, 24 hr and 7 days the percentage of the total cells rosetting with EA and EAC in-

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creased to almost 100 per cent.; the percentage of adherent cells identified as macrophages paralleled the increase in the percentage of cells showing EA and EAC receptors. In these experiments, again, there was a decrease in the total cells rosetting with EAC but not EA after treatment with BCG. This was seen best in cells obtained from younger (350 g) rather than older (500 g) animals. 1OC

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FIG. 7.-The percentage of adherent non-elicited macrophages staining with trypan blue and ethidium bromide in the presence of latex 1 mg/ml (xx), BCG at 5 mg/ml(O---O) and with no addition (0-e).

4. Changes in membrane permeability to trypan blue and ethidium bromide Changes in cell membrane permeability of adherent non-elicited and paraffin-induced macrophages after treatment with latex or BCG was measured by trypan blue and ethidium bromide exclusion. The results are shown in figs. 7 and 8. Non-elicited adherent macrophages showed a small increase in stained cells over the 7-day culture period, and this was increased about 10 per cent. on addition of latex or BCG. On the other hand, treatment of paraffininduced macrophages with BCG or latex led to a large increase in stained cells over the 7-day culture period. Hence in this case, the majority of adherent macrophages treated with BCG or latex, at 10 mg/ml and 1 mg/ml respectively, stained with trypan blue and ethidium bromide.

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FIG.In.

FIG.lb. FIG. 1.-Photomicrographs showing the non-elicited adherent cell population at the beginning of the culture period (A), and after 7 days in culture in the presence of 50 per cent. guinea-pig serum (B). Note the decrease in cell number and the large increase in cell size and complexity. Leishman’s stain. Both photomicrographs x 420. [facing page 180,

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DISCUSSION

In the present study non-elicited peritoneal macrophages (NEM) have been used and the results obtained compared with paraffin-induced macrophages. The NEM freshly prepared are small immature cells which increase in size and complexity considerably over 7 days of culture. On the other hand, paraffin-induced macrophages have entered the peritoneum in response to a

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FIG.&-The percentage of adherent paraffin-induced macrophages staining with trypan blue and ethidium bromide in the presence of BCG (10 mg/ml) or latex (1 mg/ml) (x-x); BCG (Smg/ml) of latex 0.5 mg/ml) (0-0). Control cultures with no additions (0-0).

sterile irritant and are initially mature macrophages which change little during culture. The salient feature of the work reported here is the reversibIe decrease in the percentage of macrophages showing EAC receptors after ingestion of BCG, but not after ingestion of " inert " latex and zymosan particles. Thus this decreaseisnot due to phagocytosis alone. This phenomenon is also not explained by the undoubted change in cellular composition of adherent non-elicited cells which occurs, as it is also seen in paraffin-induced cells. It cannot be easily explained on the basis of cell death, because as seen in figs. 4 and 8, adherent macrophages which stain with trypan blue and ethidium bromide I. PATH.-VOL.

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also form rosettes with EA and EAC. It is interesting that such a large percentage of adherent cells stain with trypan blue and yet remain both adherent and capable of rosetting with EA and EAC. As metabolic energy is required for macrophage adherence (North, 1969) trypan blue staining of macrophages may imply not that the cells are dead but rather that they have an altered membrane permeability or even increased membrane activity. We are left with the conclusion that the uptake of BCG in some way alters the macrophage physiology so as to remove or mask one of the receptors measured, in a reversible way. Interiorisation of receptors may be a factor, as suggested by Chambers (1977). In the case of AI(OH)3-treated cells, this substance has previously been shown to be toxic to macrophages, as judged by the release of a soluble cytoplasmic enzyme, at concentrations above 100 pg/ml (Badenoch-Jones, Turk and Parker, 1978). The concentration used in this work (50 pg/ml) does not lead to release of cytoplasmic enzymes, at least over 24 hr. It does, however, cause an irreversible decrease in the number of adherent macrophages showing EA and EAC receptors (fig. 2). It is thus probable that we are measuring an early sub-lethal toxic effect of Al(OH)3 on macrophages and this contrasts with the results obtained with BCG-treated cells. Previous studies on EA (Fc) and EAC (C3) receptors of macrophages on implanted coverslips in mice (Mariano, Nikitin and Malucelli, 1976) have shown that cells with an epithelioid appearance lost their EA receptors. In the present study, NEM showed no change in EA receptors and electron microscopy of 7-day cultures (unpublished observations) showed that the cells have an appearance of very mature macrophages. Hence it appears that there is no epithelioid cell transformation in our cultures. In a study of immunological phagocytosis by cells on implanted coverslips in the guinea-pig (Mariano, Nikitin and Malucelli, 1977) it was found that macrophages at the site of delayed hypersensitivity showed an increase in EA-SRBC phagocytosis. This was thought to be mediated via a lymphokine. In the experiments described here, addition of lymphokine in a single dose did not alter EA and EAC binding, nor EA and EAC phagocytosis. It may be that a continuous supply of lymphokine is required to demonstrate an effect on macrophage receptors or receptor-mediated phagocytosis. This work was carried out as a preliminary comparison with previous work (Ridley, Ridley and Turk, 1978) on EA and EAC binding in cryostat sections of human leprosy lesions. It was found that there were no EA or EAC receptors on foamy macrophages full of M. leprae in regressing LL leprosy. This may be paralleled, in the present study, by macrophages treated with Al(OH)3 where there was a decrease in EA and EAC receptors. EA receptors were also reduced or absent in macrophages in BL or active LL leprosy, and EAC receptors were decreased or absent relative to the bacterial load of the cells. There are suggestions, in the present work, that this may be related to the decrease in EAC receptors seen in BCG treated macrophages, although in culture this was reversible. The mechanism of decrease in measured EAC receptors on treatment of

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macrophages with BCG can only be speculative. There was, however, a concomitant phagocytosis of EAC-SRBC, as shown in the table. Previous work on immunologically-specific phagocytosis has shown that this may be responsive to the intracellular cyclic 3'5' adenosine monophosphate levels (Lima et al., 1974) and can be inhibited by the glycolytic inhibitor 2-deoxyglucose (Michl, Ohlbaum and Silverstein, 1976). Thus it may be that intracellular BCG has subtle effects on cellular physiology so as to alter the expression of membrane receptors. Further work should be directed towards an investigation in this direction. SUMMARY

EA and EAC receptors have been studied on non-elicited and paraffininduced macrophages, under a variety of culture conditions in vitro, for up to 7 days. A large decrease in the number of macrophages showing EAC receptors was found after treatment of the cells with BCG, but not " inert " particles such as latex and zymosan. This was reversible by 7 days. In the presence of a toxic material, Al(OH)3, both EA and EAC receptors were partially lost. The results obtained have been related to previous results obtained with cryostat sections of human leprosy skin lesions. We thank D r Dennis Ridley for encouragement, LEPRA and the Hospital for Tropical Disease (Special Fund) for financial support, and Dr R. A. Wolstencroft, Kennedy Institute of Rheumatology, London, for the sample of guinea-pig lymphokine.

REFERENCES BADENOCH-JONES, P., TURK,J. L. AND PARKER,D. 1978. The effects of some aluminium and zirconium compounds on guinea-pig peritoneal macrophages and skin fibroblasts in culture. J . Path., 124., 51. BERKEN, A., AND BENACERRAF, B. 1966. Properties of antibodies cytophilic for macrophages. J. exp. Med., 123., 119 CHAMBERS, T. D. 1977. The mechanism of fusion of hamster macrophages induced by anti-macrophage serum. J. Path. 122, 163. JONDAL,M., HOLM,G., AND WICZELL, H. 1972. Surface markers on T and B lymphocytes. I. A large subpopulation of lymphocytes forming non-immune rosettes with sheep red blood cells, J. exp. Med., 136, 202. LAY,W. H., AND NUSSENWEIG, V. 1968. Receptors for complement on leucocytes. J. exp. Med., 128, 991. LEU,R. W., EDDLESTON, A. L. W. F., HADDEN, J. W., AND GOOD,R. A. 1972. Mechanisms of action of migration inhibitory factor. J. exp. Med., 136, 589. LIMA,A. O., JAVIERRE, M. A., Dras DASILVA, W., AND SETTE CAMORA,D. 1974. Immunological phagocytosis: the effect of drugs on phosphodisterase activity. Experientia, 30, 945. MARIANO, M., NIKITIN,T., AND MALUCELLI, B. E. 1976. Immunological and non-immunological phagocytosis by inflammatory macrophages, epithelioid cells and macrophage polykaryons from foreign body granulomata. J. Path., 120, 151. MARIANO, M., NIKITIN,T., AND MALUCELLI, B. E. 1977. Phagocytic potential of macrophages from within delayed hypersensitivity-mediated granulomata. J. Path., 123, 27 MICHL,J., OHLBAUM, D. J., AND SILVERSTEIN, S. C. 1976. 2-deoxyglucose selectively inhibits Fc and complement receptor-mediated phagocytosis in mouse peritoneal macrophages. J. exp. Med., 144, 1465.

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NORTH,R. J. 1969. The additive effect on the spreading of guinea-pig macrophages of exogenous ATP and a surface coated with AglAb molecules. Exp. Cell Res., 54, 267. OLIVER,J. M., ZURIER,R. B., AND BERLIN,R. 1975. Concanavalin A cap formation on polymorphonuclear leucocytes of normal and beige mice. Nature, 253, 471. M. 1969. Uptake of aldehyde-treated erythrocytes by L2 cells. Inhibition RABINOVITCH, of anti-red cell antibody by coating the erythrocyte with purified proteins. Exp. Cell Res., 54, 210. REMOLD, H. G. 1973. Requirement of a-L-fucose on the macrophage membrane receptor for MIF. J. exp. Med., 138, 1065. RIDLEY,M., RIDLEY, D. S. AND TURK,J. L. 1978. Suface markers on lymphocytes and cells of the mononuclear phagocyte series in skin sections in leprosy. J. Path., 125, 91. R. A., AND COHN,Z. A. 1972. The interactions of particulate Horseradish PeroxSTEINMAN, idase (HRP)-anti HRP immune complexes with mouse peritoneal macrophages in vitro. J. Cell Biol.,55, 616.

In-vitro modification of membrane receptors on cells of the mononuclear phagocyte system.

IN-VITRO MODIFICATION O F MEMBRANE RECEPTORS O N CELLS O F T H E MONONUCLEAR PHAGOCYTE SYSTEM MARIANRIDLEY,J. L. TURKAND P. BADENOCH-JONES Department...
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