The effects of malnutrition on murine peritoneal macrophages

EXPERIMENTAL

AND

MOLECULAR

PATHOLOGY

The Effects of Malnutrition

49, 161-170 (1988)

on Murine Peritoneal

Macrophages

J. M. PAPADIMITRIOU AND IVONNE VAN BRUGGEN Department

of Pathology, Received

University January

of Western

Australia,

12, 1988, and in revised

Nedlands, form

April

6009,

Western

Australia

19, 1988

Differences were detected between peritoneal macrophages (both resident and elicited) from mice on a low protein diet and from normal animals. The concentration of resident peritoneal macrophages was lower in animals on low protein diets than in normal controls. Although total protein (and therefore cell mass) of resident macrophages from malnourished mice was increased, their contents of thiamine pyrophosphatase, succinate dehydrogenase, and non-specific esterase were disproportionately reduced. In addition they did not ingest as many ghrtaraldehyde-fixed sheep erythrocytes or attach to as many adherent C3b sensitized sheep red blood cells as those from normal animals, although reduction of nitroblue tetrazolium was unaffected. Initially (24 hr after thioglycollate), elicited macrophages from malnourished mice did not divide as frequently as those from normal mice but by 48 hr the differences were insignificant. The elicited macrophage possessed lower levels of total protein (indicating a reduced cell mass); the levels of acid phosphatase, thiamine pyrophosphatase, succinate dehydrogenase, and nonspecific esterase and nitroblue reducing activity were also proportionately reduced. They ingested fewer ghrtaraldehyde-fixed erythrocytes and reacted with fewer C3b sensitised sheep red blood cells than those from normal mice; ingestion of IgG-coated sheep erythrocytes, on the other hand, was somewhat increased. These abnormalities may influence adversely the efftciency of early phlogistic responses and favor the establishment of infection in malnourished animals. o 1988 Academic press, I~C.

INTRODUCTION It has been known for many years that the malnourished are more susceptible to infection than those on nutritionally adequate diets (Scrimshaw et al., 1959; Gross and Newberne, 1980) and many clinical and animal investigations have supported such a view (Gross and Newberne, 1980). Generally, under conditions of malnutrition, various functions of the immune system have been shown to be impaired. Depression of cell-mediated immunity with impairment of T-cell function is common (Gross and Newberne, 1980; Narayanan et al., 1977; Rose et al., 1982), while humoral responses, the complement system, and phagocytic functions are also adversely affected (Passwell et al., 1974; Price and Bell, 1975; Coovadia and Soothill, 1976; Gross and Newbeme, 1980). Mice fed diets low in protein demonstrate impaired clearance of materials injected intravenously (Gautam er al., 1975; Passwell et al., 1974; Price and Bell, 1975; Coovadia and Soothill, 1976). Studies such as these suggest that the function of mononuclear phagocytes in such malnourished animals may be impaired and reduction in the efficiency of phagocytosis has been postulated. However phagocytosis by macrophages from malnourished animals has been shown by some investigators to be unaffected, Keusch et al., 1977), although impaired chemotaxis (Keusch et al., 1977), defective candicidal and bactericidal activities (Douglas and Schopper, 1976; Keusch et al., 1977), abnormal metabolic functions (Axelrod, 1976), and reduced capacity to trigger proliferation of antigen primed T-cells in vitro have been observed (Gross and Newberne, 1980; Rose et al., 1982). In the present investigation the cytochemical characteristics of murine resident and elicited peritoneal macrophages from animals on high and low protein diets 161 0014-4800/88 $3.00 Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

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were assessed. It was postulated that low protein diets may adversely affect the synthetic rates of various proteins of macrophages and their precursors. In order to facilitate detection of affected macrophages, cytophotometry was chosen because it permits the recording of the variation of the levels of selected material in individual cells, distinct from the general statistical impressions offered by biochemical techniques. Reduction of nitroblue tetrazolium was used as a measure of superoxide release during an oxidative burst and the amount of reaction product was again measured by cytophotometry. In addition, the concentration of Fc and C3b receptors, the phagocytosis of glutaraldehyde-fixed sheep red blood cells, the uptake of radioactive uridine and thymidine, and the rate of fusion of macrophages were also assessed. MATERIALS AND METHODS Animals. Specific pathogen-free (SPF) Balb/C mice (Animal Resources Centre, Murdoch University) of both sexes were reared with their mothers under SPF conditions until 18 days of age during which time they had access to a normal diet. After that they were separated from their mothers, randomly divided into two groups, and placed respectively on high and low protein diets. Body weights were recorded weekly and animals were used 2 weeks after starting a particular diet. Approximately equal numbers of male and female mice were used in all experiments. Diets. Diets containing 20% and 4% protein were prepared according to the formulation published by Bell et al. (1976). The low protein diet consisted of 56% wheat starch, 20% sucrose, 10% peanut oil, 4% agar, 4% egg albumin (E. S. Lawrence & Co.), 4% salt mixture, 1.5% cod liver oil, and 0.5% wheat germ oil. The salt mixture contained 335 g NaCl, 645 g K,HPOI, 190 g CaHPO, * 2H20, 99 g MgSO,, 600 g CaCO,, 1.6 g KI, 7 g MnSO,, 0.4 g Cu S04, 0.4 g Al,(SO& * K,S04, 0.5 g NaF, 0.5 g ZnCl,, and 55 g ferric citrate. Vitamins in the following aqueous solution were added to the mixed diet at a rate of 25 ml/kg: 20 mg thiamine, 28 mg riboflavin, 24 mg niacin, 24 mg pyridoxine, 120 mg Ca pantothenate, 8 g choline, 8 mg folic acid, 8.8 mg biotin, 2 mg vitamin D, 0.3 mg cyanocobalamin, and H,O to 100 ml. The normal diet was similar to the above formulation except it contained 40% starch and 20% egg albumin. Diets and water were given ad libitum. Macrophages. Resident peritoneal macrophages were obtained by lavaging the peritoneal cavities of mice with 2 ml of Minimal essential medium (MEM). Elicited macrophages were collected similarly, 24 and 48 hr after the intraperitoneal injection of thioglycollate broth; to correct for weight differences, 0.5 ml/animal was given to mice on a high protein diet and 0.25 ml/animal to mice on a low protein diet. In all instances care was taken that most of the injected lavaging fluid was collected. Total cell counts were calculated after the concentration of cells in the fluids was assessed by mixing 1 vol of lavage fluid to 2 vol of white cell counting fluid and counting the cells in a hemocytometer chamber. Cell samples from the lavages were cytocentrifuged onto glass slides and stained with Giemsa, and differential counts were performed. The remainder of the cells in the lavages were placed in MEM containing 10% fetal calf serum and cells were allowed to settle for 2 hr at 37°C. After thorough washing the attached macrophages were subjected to various procedures. Unless otherwise stated macrophages from at least five mice were pooled for the various assays. In studies on elicited mac-

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rophages exudates 48 hr after injections of thioglycollate were employed unless indicated to the contrary. Fc receptors. Sheep red blood cells (SRBC) were sensitized by incubating the erythrocytes with 0.5 x the hemolytic dose of the IgG fraction of rabbit anti-SRBC hemolysin (CSL, Sussex, England) at 37°C for 30 min. Glass adherent macrophages were then presented with a 2% concentration of these cells suspended in MEM for 1 h at 37°C. The number of SRBC per macrophage was then assessed after staining with Gram’s Twort reagent. C3b receptors. SRBC were coated with IgM by incubating them with 0.5~ the hemolytic dose of the IgM fraction of rabbit anti-SRBC hemolysin for 15 min at 37°C. After washing with saline, equal volumes of these SRBC and fresh mouse serum (as a source of complement) were incubated for 10 min at 37°C and then washed twice in saline. Glass adherent macrophages were then exposed to 0.2% of these SRBC in MEM for 15 min at 37°C. The number of erythrocytes surrounding each macrophage were then counted after staining. Receptors for glutaraldehyde-fixed SRBC. SRBC fixed in 2.5% glutaraldehyde for 48 hr were washed in saline, suspended in MEM containing 10% fetal calf serum, and presented to glass-adherent macrophages at a final concentration of 0.2% for 15 min at 37°C. The number of SRBC surrounding single macrophages were enumerated after staining. Multinucleate giant cells. Macrophage syncytia were produced by implanting discs of melinex for 11 days in the subcutaneous tissues of mice. Implantation was performed at the time when the mice were placed on their respective diets. Fusion indices were calculated according to the method of Papadimitriou et al. (1973). Labeling with tritiated thymidine. The mice were given intravenously 4 kCi/g body wt of tritiated thymidine, and then sacrificed 30 min after administration. Peritoneal washings were collected, settled on subbed glass slides for I hr, washed thoroughly, fixed in methanol at 4°C for 10 min, and air-dried. They were then coated with L4 photographic emulsion (Ilford, London) and incubated at 4°C for 4 weeks. They were then developed and lightly stained with hematoxylin, and the concentration of labeled cells was determined. Labeling with tritiated uridine. Macrophages settled on glass coverslips and cultured for 1 hr were exposed to 0.5 &i/ml tritiated uridine in MEM containing 10% fetal calf serum for 30 min. After washing with MEM autoradiographs were prepared as indicated above. The density of labeling was cytophotometrically assessed using monochromatic light (480-nm wavelength). Cytochemistry. The Feulgen technique as employed by Duijndam and van Duijn (1973) was used for the demonstration of DNA. Total protein was assessed with naphthol yellow S (Chroma/Gessel-Schmid Gmbh and Co., Stuttgart, Germany) using the technique of Tas et al. (1974). Succinate dehydrogenase activity was detected with nitroblue tetrazolium (Sigma, St. Louis, MO) using the method of Kugler and Wrobel (1978), while the procedure suggested by Pearse (1972) was employed for the detection of lactate dehydrogenase activity. Acid phosphatase activity was demonstrated using AS-B1 phosphate (Sigma) and hexazop-rosaniline as recommended by Lojda et al. (1967). Nonspecific esterase activity was detected by the technique suggested by Pearse (1972) using a-naphthylacetate (Sigma) and hexazo-p-rosaniline prepared according to Lojda et al. (1964); the gallocyanin (National Aniline Division, New York) technique was employed for the demonstration of RNA (Einarson, 1951), while thiamine pyrophosphatase

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activity was demonstrated by the method of Allen and Slater (1961). All preparations were mounted in a mixture of 15.0 g Fluoromount (Gurr, Hophin and Williams, Essex, England) and 1.08 g Cargille oil (Cargille Laboratory, Cedar Grove, New York). Nitrublue tetruzolium (NBT) reduction. Phorbol myristate (Sigma) at a final concentration of 125 mg/ml was used as a trigger of the oxidative burst and then reduction of NBT (Sigma, St. Louis, USA) was induced using the method of Rook et al. (1985). The amount of reaction product in single cells was determined by scanning cytophotometry. Cytophotometry. Scanning cytophotometry was performed on a Zeiss cytoscan microcope interfaced to a PDPI l/O3 computer. The cells were scanned using a step size of 0.5 ym; scanning and computation were achieved with the HIDACSYS programs of van der Ploeg et al. (1974). The wavelength of monochromatic light used was as follows: 561 nm for Feulgen-DNA, 430 nm for cells stained with naphthol yellow, 517 nm for cells in which acid phosphatase activity was demonstrated, 580 nm for cells in which nonspecific esterase activity was demonstrated, 545 nm for cells in which nitroblue tetrazolium reduction occurred, and 570 nm for gallocyanin-stained cells. A minimum of 250 cells was measured on each preparation. RESULTS Animals. As expected, a significant difference in body weight was present between mice on a diet containing 20% protein and those on a 4% protein diet. After 2 weeks on the appropriate diets, the mean weight of mice on a 20% protein diet was 11.68 * 1.68 g, while that of mice on a 4% protein diet was 5.47 2 0.77 g. Mice fed the 4% protein diet consumed less food per day than mice given the 20% protein diet, although they consumed more food per gram body weight than the control group. (Price et al. (1975) using the same diets have observed that when pair-fed mice were given the amount of food consumed by mice on low protein diets, they also exhibited a significant reduction in body weight.) Peritoneal macrophages. Generally the yield of resident peritoneal macrophages from the unstimulated peritoneal cavity of mice on a high protein diet (11 mice) was significantly higher (P < 0.01) than that from those on a low protein diet (7 mice); the concentration in the group on a high protein diet was 8.1 4 2.3 x lO”/g body wt and 5.7 +- 1.3 X lO”/g body wt for mice on a low protein diet. On the other hand when the peritoneal cavity was stimulated with thioglycollate and the macrophages were collected 24 hr afterward, no significant differences were detected between the two groups; the concentration of macrophages from mice on a high protein diet (7 mice) was 2.3 2 0.5 X 105/g body wt and it was 2.4 -t 1.2 x lo5 g body wt for mice on a low protein diet (9 mice). Quantitative cytochemistry of resident peritoneal macrophages (RPM). Quantitation of Feulgen-DNA staining of RPM from mice on high and low protein diets did not reveal any differences between the two groups. Estimation of total protein on the other hand, showed that RPM from mice on a low protein diet possessed more protein per cell than those from the high protein group (Table I). Total cellular RNA did not differ between the two groups nor did the total succinate dehydrogenase activity (Table I). Acid phosphatase lactate dehydrogenase and nitroblue reducing activities, however, were greater in RPM from mice on a low

MACROPHAGES

Comparison of the Cytophotometric

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MICE

TABLE I Assays in Resident Peritoneal Macrophages from Normal (N) and Malnourished (M) Mice Absorbance/cell”

Total protein N M RNA N M Nonspecific esterase N M Acid phosphatase N M Thiamine pyrophosphatase N M Lactate dehydrogenase N M Succinate dehydrogenase N M NBTb reduction N M

Mean

SD

f value

P

1,712 2,485

315 115

11.48404

<0.0005

1.4

6,294 6,101

1450

2.15083

NSD

1.1

8,141 6,565

2121 2343

8.86836

<0.0005

0.8

3,943 8,103

3809

11.99458

-Go.0005

2.2

3,194

112 621

18.001108

<0.0005

0.1

3.63446

<0.0005

1.2

2.54299

NSD

2,512

Ratio M:N

1550

4150

9,564

4150

11,130

4460

6,591 6,066

2080

6,411 8,352

3094 3121

0.9

2160 6.11281

<0.005

1.3

n Absorbance is expressed in arbitrary units. b NBT, nitroblue tetrazolium.

protein diet than in RPM from mice on a high protein diet (Table I), while thiamine pyrophosphatase and nonspecific esterase activities (Table I) were greater in RPM from mice on a high protein diet. The higher content of total protein of RPM from malnourished animals indicates that the cells have a greater mass than those from normal mice. The ratio of the protein content of RPM from the two groups of mice, however, does not match the ratios for the various enzymatic activities of RPM in the two groups. With the exception of the ratio for acid phosphatase activities, which is higher, and nitroblue tetrazolium reduction and lactate dehydrogenase activities, which are similar, all other ratios are lower than that for total protein. In other words if cellular mass is taken into consideration, the activities investigated, with the exception of acid phosphatase, are either unaffected or decreased in RPM from mice on a low protein diet. Quantitative cytochemistry of elicited peritoneal macrophages (EPM). Again comparisons of the amounts of Feulgen-DNA in EPM from mice on the high and low protein diets did not reveal any significant difference. On the other hand the levels of total protein, acid phosphatase, thiamine pyrophosphatase, succinate dehydrogenase, nonspecific esterase, lactate dehydrogenase, and nitroblue reduc-

166

PAPADIMITRIOU

Comparison of the Cytophotometric

AND VAN BRUGGEN

TABLE II Assays in Elicited Peritoneal Macrophages” and Malnourished (M) Mice

from Normal (N)

-

Absorbancelcellb

Total protein N M RNA N M Nonspecific esterase N M Acid phosphatase N M Thiamine pyrophosphatase N M Lactate dehydrogenase M Succinate dehydrogenase N M NBTC reduction N M

Mean

SD

t value

P

2,101 1,605

716 426

8.45790


0.8

15,191 14,241

6326 7168

1.40917

NSD

0.9

3,634 3,111

1375 1123

4.23666


0.9

19,814 16,393

5375 4638

6.84013

<0.0005

0.8

6,663 2,554 10,225 8,844

4506 1357 4230 3803

12.03619

<0.0005

0.4

3.33351


0.9

8,352 5,970

3579 2576

7.76982

<0.0005

0.7

10,960 9,408

3233 2464

5.95149

<0.0005

0.9

0 Elicited peritoneal macrophages were collected 48 hr after the intraperitoneal glycollate. b Absorbance is expressed in arbitrary units. ’ NBT, nitroblue tetrazolium.

Ratio M:N

injection of thio-

ing activities were greater in EPM from the high protein group than in EPM from the low protein group (Table II). Total cellular RNA, however, was similar in both groups. These reductions, however, are not as significant when the ratios of the various activities in EPM from the two groups are compared with that for total protein (Table II). The value of the latter indicates that EPM from malnourished mice are smaller than normal. However, with the exception of the ratio for thiamine pyrophosphatase activities, which is low, the ratios of all other activities are similar to that for total protein. In summary then, if cellular mass is considered, most activities in EPM are relatively unaffected. Tritiated thymidine and uridine labeling of resident and elicited peritoneal macrophages. After intravenous injection of tritiated thymidine 2.4 + 1.7% of RPM from mice on a high protein diet were labeled, while the concentration in similarly treated mice on a low protein diet was 1.5 f 1.0%, the difference not being statistically significant. On the other hand, 24 hr after ip thioglycollate injection 2.4 + 1.1% of EPM from mice on high protein diets and 0.28 -t- 0.12% of those from mice on a low protein diet incorporated the label, the difference being statistically significant (P < 0.0005). By 48 hr after ip thioglycollate injection, how-

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ever, the concentration of labeled cells from both groups of mice was similarthe proportion of labeled macrophages from normal mice was 1.8 + 2.3%, while that from animals on a low protein diet had risen to 1.2 +- 1.4%. Uridine labeling followed by cytophotometry of the labeled cells did not demonstrate any significant difference between RPM from mice on a high protein diet and those on a low protein diet. Similar results were obtained when EPM from these two groups of mice were compared. Fc receptors, C3b receptors, and receptors for glutaraldehyde-fixed erythrocytes. Enumeration of attached or ingested appropriately treated

sheep

erythrocytes onto RPM was used as a measure of various receptors. No major differences in the distribution of Fc receptors were detected between RPM from mice on a high protein diet and those from mice on a low protein diet (Table III). On the other hand, receptors for C3b and for glutaraldehyde-fixed sheep red blood cells were more frequent on the surface of RPM from mice on a high protein diet than on the surface of those from mice on a low protein diet (Table III). Similar results were obtained when EPM frqm mice on high protein and low protein diets were compared (Table IV), except that somewhat more Fc receptors were present on the surface of EPM from malnourished mice than on the surface of those from mice on a high protein diet. Macrophagefusion. Assessment of the efficiency of fusion of macrophages on subcutaneously implanted melinex discs did not reveal any significant differences between those from mice on low protein diets and those from mice receiving high protein diets. The fusion index (Papadimitriou et al., 1973) of macrophages adhering to implanted melinex discs in malnourished mice was 47.6 -+ 13.1%, while that in normal mice was 38.4 k 14.7%. DISCUSSION The results of these investigations indicate that distinct differences exist between peritoneal macrophages from mice on low protein diets and from those on high protein diets. In addition to numerical and cytochemical differences, functional anomalies were also detected which were present in both resident and elicited peritoneal macrophages. Because malnourished mice are smaller than those receiving a normal diet, the TABLE III Assessment of Various Receptors on Resident Peritoneal Macrophages from Normal (N) and Malnourished (M) Mice Number of erythrocyteskell Receptor Fc receptor N M C3b receptor N M

Receptor for fmed SRBC” N M n SRBC, sheep red blood cells.

Mean

SD

t value

6.8 7.8

6.7 8.3

1.62107

28.5 25.6

12.3 13.1

2.79061

9.8 4.0

7.6 3.2

12.16211

P NSD

co.005

<0.0005

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PAPADIMITRIOU

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TABLE IV Assessment of Various Receptors on Elicited Peritoneal Macrophages from Normal (N) and Malnourished (M) Mice” Number of erythrocyteskell Receptor Fc receptor N M C3b receptor N M Receptor for fixed SRBC** N M

Mean

SD

t value

P

12.2 15.6

9.7 10.4

4.16630


22.3 17.3

11.8 12.7

4.98726


17.1 14.9

6.8 8.7

3.44511


n Elicited peritoneal macrophages were collected 48 hr after an intraperitoneal glycollate. b SRBC, sheep red blood cells.

injection of thio-

numbers of both resident or exudate peritoneal macrophages in each mouse were corrected by expressing them in terms of body weight. When expressed this way it became clear that mice on a low protein diet have fewer resident peritoneal macrophages than those on a normal diet. These differences, however, disappear when the peritoneum is inflamed, indicating that the intluence of diet on the initial monocytic exudation after a phlogistic stimulus is similar in both malnourished and normal mice if expressed in terms of weight. It seems, therefore, that the differences that have been reported previously (Rose et al., 1982; Price and Bell, 1975), when total numbers of exudate macrophages in the peritoneal cavity of normal and protein-deficient mice were being compared, can be accounted by the differences in body mass. Pulse labeling with tritiated thymidine demonstrated that the turnover of resident peritoneal macrophages was similar in both the high and low protein groups. A similar assessment on elicited macrophages showed that initially (24 hr after thioglycollate injection) the rate of division is higher in those from mice on a high protein diet than in those from mice on a low protein diet, indicating that division of newly emigrated monocytes in protein malnourished animals will be inefficient. By 48 hr after thioglycollate injection, however, these differences became insignificant . The uptake of tritiated uridine was not significantly different in the two groups of mice, indicating that the rate of transcription in individual cells is not greatly altered in macrophages from protein-deficient mice. This together with the lack of variation of cytochemically detectable cellular RNA in individual macrophages indicates that RNA metabolism is unaffected. Measurement of total protein shows that RPM from malnourished mice have a greater cell mass than those from normal mice. Most of the enzymatic activities that were investigated had not, however, increased proportionately, indicating that these cells are to some degree functionally inefficient. Acid phosphatase activity on the other hand increased disproportionately. The reason for this is unclear; possibly the rate of transcription for this enzyme is preferential or a consequence of such macrophages entering a predominantly catabolic phase. Re-

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duction of nitroblue tetrazolium is largely unaffected, hinting that at a cellular level the microbicidal properties of RPM are generally unaltered by malnutrition. Estimates of total cell protein on EPM on the other hand indicate that those from malnourished mice have a smaller mass than those from normal animals. Apart from the levels of thiamine pyrophosphatase, however, the other activities are not disproportionately affected. The reasons for the low levels of thiamine pyrophosphatase are unclear. Functional aberrations were also detected when resident and elicited peritoneal macrophages were tested for the presence of surface receptors that are involved in adhesion and endocytosis. Attachment of sheep erythrocytes dependent on the C3b receptor or that responsible for ingestion of glutaraldehyde-fixed sheep erythrocytes was reduced in both RPM and EPM from mice on low protein diets. Presumably this reflects a reduction in the turnover of some receptor proteins in macrophages from malnourished mice. These findings support reports that at least some endocytic functions are impaired by protein malnutrition (Passwell et al., 1974; Price and Bell, 1975; Coovadia and Soothill, 1976; La Via et al., 1956), as well as those that have shown defects of macrophage chemotaxis (Gross and Newberne, 1980; Rose et al., 1982). The increased ingestion of IgG-coated sheep erythrocytes by EPM is, however, unexplained; somewhat similar results have also been observed by Cooper et al. (1974) and Hamm and Winick (1984) in their studies with protein-deficient animals. Generally then malnourished mice possess fewer but larger RPM than normal animals; these are, however, metabolically and functionally somewhat inefficient. Elicited macrophages from malnourished mice on the other hand, are smaller than normal and although their metabolic and functional efficiency is only minimally disturbed, their mitotic rate is, at least initially, significantly reduced. These anomalies may intluence adversely the efficiency of the early phlogistic responses and favor the establishment of infection. ACKNOWLEDGMENTS This work was supported by a grant from the National Health and Medical Research Council of Australia.

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