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Articles

Overabundance of CD45RA⁺ (Quiescent-Phenotype) Cells within the Involuted CD4⁺ T-Cell Population Follows Initiation of Immune Depression in Energy-Deficient Weanling Mice and Reflects Involution Exclusive to the CD45RA^- Subset¹

Department of Human Biology and Nutritional Sciences, University of Guelph, Guelph, ON, Canada N1G 2W1 and ^* Department of Health and Nutrition, Chia-Nan University of Pharmacy and Science, Jen-Ter Hsiang, Tainan 71710, Taiwan

²To whom correspondence should be addressed. E-mail: wwoodwar{at}uoguelph.ca.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Previous studies have identified an overabundance of quiescent-phenotype (CD45RA⁺) CD4⁺ T cells throughout the lymphoid system of weanling mice at an advanced stage of food intake restriction mimicking marasmus. The objective of this investigation was to determine the timing of this phenomenon relative to the development of depression in cell-mediated immune competence. Two experiments were conducted in which male and female weanling C57BL/6J mice, initially 19 d of age, either were permitted free access to a complete purified diet or were subjected to restricted intake of this diet, producing loss of 1.5–2% of initial body weight daily. In the first experiment, feeding periods of 3, 6, 9, 12 and 14 d were examined, and a zero-time control group (19 d old) was also included. Expression of CD45RA was assessed by flow cytometry in CD4⁺ T cells from the blood, spleen and mesenteric lymph nodes. Despite reduction in CD4⁺ T-cell numbers, evident in all three lymphoid compartments of the malnourished mice by d 6, energy-restricted mice maintained the numbers of CD4⁺CD45RA⁺ T cells at the level found in the zero-time control group. Consequently, the malnourished group exhibited a high percentage of CD4⁺ T cells expressing CD45RA by d 9 in the blood and mesenteric nodes and by d 12 in the spleen. In the second study, malnourished and age-matched control groups were sensitized to sheep red blood cells on d 3 and energy-restricted mice exhibited depression in the delayed hypersensitivity response to this antigen when assessed on d 9 after challenge 24 h previously. Energy deficiency pathology includes a shift toward CD4⁺ T cell quiescence that may contribute to ongoing immunodepression without being involved in its initiation. Remarkably, this imbalance develops because involution of the CD4⁺ subset in the energy-deficient mice is confined to the CD45RA^- population.

KEY WORDS: • mice • T cell • blood • spleen • lymph node

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Lymphoid involution is characteristic of wasting, prepubescent protein-energy malnutrition (PEM),³ and is generally considered to contribute to the depression in thymus (T)-dependent immunocompetence that is a feature of this disease (1) . At the same time, intervention studies using the hormone, triiodothyronine, reveal that depression in T-dependent immunity can be prevented (2 3 4) or even reversed (5) , despite unabated and profound lymphoid atrophy in which cellular losses exceed 90%. It is reasonable, therefore, to pursue other features of the immunobiology of PEM, whether deficiency of protein or of energy, that may prove more basic than lymphoid involution to depressed acquired immunity in wasting disease.

Imbalances among critical subsets of T cells may contribute to PEM-associated immunodepression (1) . This proposition has received support recently from the study of wasting protein and energy deficiencies in weanling mice. In these metabolically dissimilar pathologies, an overabundance of CD45RA⁺ T cells is reported within the CD4⁺ subset throughout the involuted lymphoid system (6 ,7) . The CD4⁺ CD45RA⁺ T cell is quiescent relative to its CD45RA^- counterpart in terms of proliferative and cytokine responses to stimulation through the T-cell receptor (8 9 10 11 12) . In fact, expression of CD45RA appears to identify the extreme of hyporesponsiveness within the quiescence-activation spectrum of CD4⁺ T cells (11) . Thus, an imbalance favoring quiescence within the CD4⁺ T-cell subset may contribute to depressed T-dependent immunocompetence in weanling PEM induced by deficit of either protein or energy.

It is important to identify the time-kinetics of immunologic change in PEM. In this connection, the overabundance of CD45RA⁺CD4⁺ T cells is confined to the latter stages of weight loss pathology in a weanling murine model that mimics incipient kwashiorkor and in which nitrogen is the limiting dietary factor (7) . Thus, in protein deficiency pathology, depression in T-dependent immune competence precedes the quiescence-shift within the CD4⁺ subset, and this imbalance phenomenon may contribute to ongoing immune depression without being involved in its initiation (7) . However, weight loss through energy restriction produces a marasmic condition in weanling mice that is metabolically distinct from wasting protein deficiency (2 ,13 ,14) . No information is available regarding the time-kinetics whereby an overabundance of quiescent-phenotype CD4⁺ T cells develops in wasting energy deficiency. Therefore, the objective of the present investigation was to determine the timing of the shift toward expression of CD45RA within the CD4⁺ T-cell subset relative to the appearance of depressed cell-mediated immune competence during the progression of marasmic weight loss pathology in weanling mice.

	MATERIALS AND METHODS

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Animals and feeding protocols.

Mice were used from an in-house breeding colony (Department of Human Biology and Nutritional Sciences, University of Guelph, Canada) of the C57BL/6J strain. The mice from which the colony was derived were purchased several years earlier from the Jackson Laboratory (Bar Harbor, ME). Male and female mice were weaned at 18 d of age, acclimated for 1 d to a complete purified diet and allocated subsequently to experimental groups. Malnourished groups were fed restricted quantities of the complete diet, and age-matched control groups were given free access to the same diet. The quantity of diet provided to each malnourished mouse was determined daily by means of equations derived in this laboratory (15) relating ad libitum food intake (g food/g body) to chronological age in the weanling mouse. Thus, the percentage of predicted ad libitum intake provided to each mouse (generally between 40 and 60%) was determined on the basis of the unique rate of weight loss of the mouse. This strategy permitted a high degree of uniformity among mice in achieving a loss of 1.5–2% of initial body weight/d as described previously (6 ,7) . All mice had unimpeded access to clean tap water at all times. Mice were killed by cervical dislocation while anesthetized with Metofane (Pitman-Moore, Mississauga, Canada).

Mice were housed individually in plastic cages with stainless steel tops. The animal facility was a windowless room maintained at 25–27°C and at a relative humidity of 60–70%; the room was supplied with fluorescent light daily between 0800 and 2200 h. The experiments, procedures and housing conditions (a conventional facility) were approved by the Animal Care Committee of the University of Guelph and were in accordance with the recommendations of the Canadian Council on Animal Care.

Diets.

The complete purified diet has been described in detail (16) and meets or exceeds current standards of the NRC (17) for laboratory mice. A typical proximate analysis for this diet is 92.3% dry matter, 18.8% crude protein, 8.1% ether extract, 2.6% ash, 3.1% crude fiber and 17.0 kJ/g gross energy (4) . Coprophagy was permitted in the present investigation as in previous studies of T cells and immune competence in the same experimental system (2 ,6 ,7) .

Experiment 1.

Malnourished and age-matched control groups were examined after feeding periods of 3, 6, 9, 12 or 14 d. In addition, a zero-time control group was included in the design; therefore, they were examined at 19 d of age immediately after acclimation. Because of low cell numbers, pooling was necessary for all malnourished groups (3 mice/sample) as well as for the zero-time control group and the 3- and 6-d age-matched control groups (2 mice/sample). Sample sizes of 6 (zero-time control group) and 8 (all other groups) were studied. Males and females were used in similar numbers. Each pooled sample comprised one gender only and constituted a single degree of freedom for the purpose of statistical analysis.

Experiment 2.

Previous results showed that the food intake restriction protocol used in this investigation produces depression in T-dependent immune competence that is apparent in C57BL/6J weanlings when sensitized at d 9 of weight loss and assessed 5 d later (6) . However, no information was available regarding earlier stages of weight loss in animals subjected to this form of malnutrition. Therefore, in view of the results of the first experiment of this investigation, a supplementary experiment was performed. The objective was to determine cell-mediated immune competence in the early stages of weight loss imposed according to the food intake restriction protocol used in this investigation. Sample sizes of 8 (immunized mice in malnourished and age-matched control groups, and shams in the age-matched control group) and 9 (malnourished group, shams) were studied, and immune competence was assessed on d 9 of food intake restriction after sensitization and challenge on d 3 and 8, respectively. As in the first experiment, each group included males and females in approximately equal numbers.

Procedures to obtain blood samples, spleen and lymph nodes.

Procedures to obtain blood, spleen and nodal tissue were described previously (7 ,14) . Briefly, blood samples from the orbital plexus of mice anesthetized with Metofane (Pitman-Moore) were collected into heparinized microcentrifuge tubes. Mice were then killed by cervical dislocation, and the spleen and mesenteric nodes were removed aseptically into RPMI 1640 medium (Flow Laboratories, Mississauga, Canada) containing 5% heat-inactivated fetal calf serum (Sigma Chemical, St. Louis, MO) and 1 mmol/L HEPES (ICN Biomedicals Canada, St. Laurent, Canada).

Identification of cellular subsets by flow cytometry.

Single-cell suspensions of mononuclear cells for analysis by flow cytometry were prepared from the spleen, mesenteric lymph nodes and blood as described previously (7 ,14) . Viability before staining was determined by eosin Y exclusion and always exceeded 95%. Cellular subsets were identified by means of a Coulter Epics XL-MCL flow cytometer (Beckman Coulter, Mississauga, Canada) equipped with version 1.5 (1993) software. Staining reagents included phycoerythrin-R-conjugated anti-CD4 (YTS 191.1; Cedarlane Laboratories, Hornby, Canada), unconjugated anti-CD45RA (RA3–2C2/1) produced and purified in this laboratory as described elsewhere (6) and fluorescein isothiocyanate-conjugated immunoglobulin G F(ab)₂ fragment of affinity-purified goat anti-rat µ heavy chain (Bio/Can Scientific, Mississauga, Canada). Details pertaining to these reagents and their use are provided elsewhere, as are details of the reagents and procedures applied for the purpose of negative control staining and Fc receptor blockade (6 ,7 ,14) . Each analysis, including those of negative control samples, was based on at least 10⁴ events after dead cells and residual erythrocytes were eliminated by gating on the basis of forward angle light scatter.

Assay of cell-mediated immune competence.

The delayed hypersensitivity response to low dose sensitization with sheep red blood cells (SRBC) was elicited as described previously (18) . Briefly, on d 3 of the experimental period, each mouse received a single 100-µL intraperitoneal injection of either pyrogen-free saline (MTC Pharmaceuticals, Cambridge, Canada) or 8 x 10⁵ SRBC (CedarLane Laboratories) in pyrogen-free saline. On d 8, each mouse was challenged by injection into the left hind footpad of 1.2 x 10⁸ SRBC in 20 µL pyrogen-free saline. The right hind footpad of each mouse received the same volume of saline carrier and served as the unchallenged foot. The response was quantified 24 h after challenge as the quotient of the maximum thickness of the challenged foot divided by the maximum thickness of the unchallenged foot.

Carcass analyses.

Carcasses were stored at -20°C for not >20 wk before analysis. Dry matter, lipid and crude protein contents were determined as described elsewhere (4 ,6 ,14) .

Statistical analysis.

The predetermined upper limit of probability for statistical significance throughout this investigation was P 0.05. For the purpose of comparison to the zero-time control group in Experiment 1, data were subjected to one-way ANOVA, followed (if justified by the resulting statistical probability value, i.e., P 0.05) by Duncan’s New Multiple Range test. For comparison of restricted and age-matched control groups in Experiment 1, data sets not including the zero-time control group were subjected to two-way ANOVA followed (if justified by the statistical probability of the diet main effect) by preplanned least squares means comparisons within time points. Similarly, two-way ANOVA and least squares means comparisons were applied to the data in Experiment 2. If the error term of a data set failed to exhibit normal distribution after application of several transformation procedures, the Kruskal-Wallis test (² approximation) was applied to Wilcoxon rank sums and, where warranted by the resulting statistical probability value (i.e., P 0.05), this analysis was followed by ² comparisons of Wilcoxon two-sample rank sums for each combination of treatment pairs.

	RESULTS

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Experiment 1: The food intake restriction protocol produced wasting disease including involution of the mononuclear cell compartments of blood, spleen and mesenteric lymph nodes.

Growth indices and mononuclear cell counts are presented in Table 1 . The wasting malnutrition produced by d 14 in the present investigation was comparable to the marasmic pathology reported previously in studies demonstrating depressed T-dependent immunocompetence in the same experimental system (2 ,5 ,6 ,14) . Thus, food intake restriction produced weight loss reaching 20% of initial body weight after 14 d, whereas mice permitted ad libitum intake gained steadily, both fat and lean tissue, and doubled their body weight within the same period of time. Loss of fat was particularly remarkable in the malnourished groups, such that the percentage of carcass lipid was low relative to age-matched and zero-time controls as early as 3 d into the weight loss protocol. Because of the extent of fat loss, the percentage of dry matter was lower in the malnourished groups than in age-matched controls at each stage of weight loss examined after d 3. However, this did not reflect edema because carcass fat-free dry matter content was not affected by diet (results not shown). Comparison of food intake–restricted groups with the zero-time control group revealed a low blood mononuclear cell count (per µL basis) by d 3 and mononuclear involution of the spleen and mesenteric nodes by d 6. In contrast, the well-nourished controls maintained the blood mononuclear cell count observed at 19 d of age and, within 3 d, exhibited increases relative to zero-time controls in the number of mononuclear cells recoverable from the spleen and mesenteric nodes.

Two-color flow cytometer histograms illustrating the expression of the surface marker CD45RA by CD4⁺ T cells of the blood, spleen and mesenteric nodes have been published previously from this laboratory for C57BL/6J weanlings subjected to the experimental protocols used in the present investigation (6 ,7) . The percentages of CD4⁺ T cells exhibiting a CD45RA⁺ phenotype within the mononuclear cell suspensions of blood, spleen and mesenteric nodes are shown in Figure 1 . No ontogenetic change was apparent in this index by comparison of the age-matched and zero-time control groups. Food intake restriction produced an increase, relative to both zero-time and age-matched controls, in the proportion of cells expressing CD45RA within the CD4⁺ subset of the blood and mesenteric nodes by d 9 and of the spleen by d 12.

View larger version (21K): [in this window] [in a new window]   Figure 1. Percentage of CD4+ T cells expressing CD45RA in mononuclear cell suspensions from the blood, spleen and mesenteric nodes of mice given free access to a complete diet for 3, 6, 9, 12 or 14 d, mice fed the complete diet in restricted daily quantities for the same periods of time to produce weight loss of 1.5–2% of initial body weight daily, and a zero-time control group representing the beginning of the experimental feeding period, i.e., mice at 19 d of age. Bars represent mean values and, for the blood and mesenteric nodes, represent squares of square root–transformed data. Sample sizes of 6 (zero-time control group) and 8 (all other groups) were studied, and pooling was necessary for recovery of sufficient cell numbers from the malnourished groups (3 mice/sample), from the zero-time controls (2 mice/sample) and from the 3- and 6-d age-matched control groups (2 mice/sample). Males and females were represented in similar numbers in all groups. Each pooled sample comprised one gender, only, and constituted a single degree of freedom for the purpose of statistical analysis. The data from males and females were combined to produce the results shown in the figure. Asterisks indicate mean values that differ from zero-time controls as determined by one-way ANOVA followed by Duncan’s New Multiple Range test (P 0.05). For this analysis, pooled SEM = 0.435, 3.339 and 0.538, respectively, for blood, spleen and mesenteric nodes. Each data set, not including the zero-time controls, was also subjected to two-way ANOVA; the main effects were diet and time, and the letter "a" indicates statistical difference from age-matched control (P 0.05) by least squares means comparison. Pooled SEM (two-way ANOVA) = 0.0237, 2.835 and 0.4475, respectively, for the blood, spleen and mesenteric nodes. For the blood, P = 0.0001 (diet), 0.0001 (time) and 0.0001 (interaction). For the spleen, P = 0.0442 (diet), 0.0003 (time) and 0.0021 (interaction). For the mesenteric nodes, P = 0.0001 (diet), 0.0018 (time) and 0.0003 (interaction).

View larger version (28K): [in this window] [in a new window]   Figure 2. Numbers of CD4+ and CD4+CD45RA+ T cells in mononuclear cell suspensions from the blood, spleen and mesenteric nodes of the groups of mice identified in the legend to Figure 1 . Points represent mean values. For CD4+CD45RA+ cells of the blood, means are squares of square root–transformed data, whereas means are antilogs of natural log–transformed data for the spleen as well as for CD4+ cells of the mesenteric nodes. Procedural details are identical to those presented in the legend to Figure 1 . Asterisks indicate mean values that differ from zero-time controls (P 0.05) according to one-way ANOVA followed by Duncan’s New Multiple Range test or, for the data pertaining to CD4+CD45RA+ cells of the mesenteric nodes, according to the Kruskal-Wallis procedure followed by 2 comparisons of Wilcoxon two-sample rank sums. For these analyses, pooled SEM = 41.5 and 0.793 (CD4+ and CD4+CD45RA+ cells of the blood), 0.255 and 0.389 (splenic CD4+ and CD4+CD45RA+ cells) and 0.275 (CD4+ cells of the mesenteric nodes), whereas Kruskal-Wallis rank sums for CD4+CD45RA+ cells of the mesenteric nodes were 67 (zero-time controls), 262, 311, 257, 364 and 147 (age-matched controls at d 3, 6, 9, 12 and 14, respectively) and 137, 53, 151, 204 and 127 (malnourished at d 3, 6, 9, 12 and 14, respectively). With the exception of data for the CD4+CD45RA+ cells of the mesenteric nodes (which did not normalize), each data set, with the zero-time control group excluded, was also analyzed by two-way ANOVA, with main effects of diet and time. The letter "a" indicates statistical difference (P 0.05) between restricted and age-matched control according to subsequent least squares means comparison or according to Wilcoxon two-sample comparisons in the case of nodal CD4+CD45RA+ cells. Pooled SEM (two-way ANOVA, CD4+ cells) = 33.15, 0.201 and 0.227, respectively, for the blood, spleen and mesenteric nodes. Pooled SEM (two-way ANOVA, CD4+CD45RA+ cells) = 0.109 and 1.57, respectively, for blood and spleen. For CD4+ blood cells, P = 0.0001 (diet), 0.1064 (time) and 0.2618 (interaction). For CD4+ spleen cells, P = 0.0001 (diet), 0.0147 (time) and 0.0001 (interaction). For CD4+ node cells, P = 0.0001 (diet), 0.0005 (time) and 0.0001 (interaction). For CD4+CD45RA+ blood cells, P = 0.9843 (diet), 0.0032 (time) and 0.0681 (interaction). For CD4+CD45RA+ spleen cells, P = 0.0001 (diet), 0.0207 (time) and 0.0526 (interaction).

Experiment 2: Depressed cell-mediated immune competence was evident in wasting animals sensitized after only 3 d of food intake restriction.

The results from the supplementary experiment are shown in Table 2 . The mice exhibited initial body weights, weight changes and food intakes that were comparable to those of the corresponding groups (age-matched controls or malnourished, d 9) in the first experiment. Therefore, the wasting disease imposed on the malnourished groups was comparable to that produced in the first study. The age-matched control mice exhibited a delayed hypersensitivity response similar in magnitude to the response reported previously for weanling C57BL/6J mice (18) . In contrast, the response of immunized malnourished mice was indistinguishable from that of sham-injected negative controls.

	DISCUSSION

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Previous studies in this laboratory have shown that the type of anti-SRBC response elicited in this investigation is neither an Arthus reaction nor a Jones-Mote type of hypersensitivity, but exhibits the characteristics of delayed hypersensitivity, i.e., a cell-mediated response (18) . The antigen-specific component of the delayed hypersensitivity response in mice is mediated exclusively by the CD4⁺ T cell (20) , and the anti-SRBC response in mice is analogous to the classical tuberculin reaction (21) . In the present investigation, antigen was administered by way of the peritoneum, thereby ensuring that the anti-SRBC response was elicited within the spleen (22) . Consequently, the measure of cell-mediated immune competence assessed in this investigation points to the CD4⁺ T-cell subset of the spleen. No malnutrition-related effect was apparent on the proportion of quiescent-phenotype CD4⁺ T cells in the spleen until d 12, i.e., a stage of wasting disease subsequent to the interval during which the anti-SRBC response was elicited and assessed. Clearly, therefore, the quiescence of the CD45RA⁺CD4⁺ T cell (9 10 11 12) is not a factor in initiating immune depression in food intake-restricted weanling mice, although a quiescence-shift within the CD4⁺ subset may contribute to ongoing immune depression in the advanced stages of weight loss pathology induced in this way. The same conclusion has been reported with reference to a model of weanling murine protein deficiency that represents incipient kwashiorkor (7) . The profound metabolic dissimilarities between the low protein model and the food intake restriction system of the present investigation have been discussed elsewhere (2 ,13 ,14) . The low protein protocol imposes weight loss as a consequence of nitrogen deficiency, i.e., through a dietary imbalance, whereas the food intake restriction system produces energy deficit without deficiency of nitrogen or other nutrients. Thus, it appears that overabundance of quiescent-phenotype CD4⁺ T cells is a basic characteristic of wasting pathologies in their advanced stages, and may be expected to contribute to the immunodepression that is a constant feature of these conditions.

Energy deficiency induced profound involution of the CD4⁺ T-cell compartment in this investigation. After 14 d of weight loss, cell numbers in this compartment of the spleen and mesenteric nodes were only 10% of the numbers found in the zero-time control group. This outcome is consistent with other reports pertaining to this model (6 ,7 ,14) and is a consistent feature of wasting PEM (1) . However, a remarkable outcome of the present investigation is that the malnutrition-induced involution of the CD4⁺ subset was confined to the CD45RA^- population, which is relatively hyperreactive (11) and enriched in memory/effector cells (12) , whereas the size of the population representing extreme quiescence among CD4⁺ T cells (11) remained unaffected into advanced weight loss. Memory/effector-phenotype T cells exhibit rapid turnover as a population (23 ,24) , but previous results show that the vulnerability of this population to malnutrition-induced involution cannot be understood simply on the basis of cellular life span, i.e., on the basis of a presumptive inability to support cellular replacement (6) . Rather, the phenomenon appears to relate more closely to cellular activation than to cellular life span, and may be discernible phenotypically only through use of a marker such as CD45RA which identifies extreme hyporesponsiveness in the quiescence-activation spectrum of the CD4⁺ T cell (6) . The astonishing resistance of the quiescent-phenotype CD4⁺ T-cell population to malnutrition-induced involution was not anticipated and deserves further study.

The blood is the main source of lymphocytes for the clinical and experimental assessment of humans. Nevertheless, indices pertaining to blood T cells cannot be presumed ipso facto to reflect the T cells within secondary lymphoid organs in which primary responses to antigen arise (25) . The results of the present investigation, however, confirm the finding reported previously (7) that the blood can provide systemically relevant information regarding the effect of advanced stages of acute weight loss on the expression of CD45RA by T cells. Further, it may now be concluded that the blood provides a window, throughout development of weight loss pathology, on this quiescence-related characteristic of CD4⁺ T cells. This is particularly interesting in view of a report that PEM produces a shift toward expression of CD45RA by blood T cells of elderly humans (26) . Clearly, the T cell quiescence-shift phenomenon is not confined to rodents, or to the prepubescent stage of life, and the possibility arises that expression of CD45RA by blood CD4⁺ T cells might prove useful as a marker in the management of human wasting pathologies.

The present investigation, taken together with a previous study of weanling protein deficiency (7) , indicates that quiescence associated with CD45RA⁺CD4⁺ T cells may contribute to immune depression during the advanced stages of weight loss in metabolically diverse wasting pathologies. It will be important to pursue the phenotypic evidence produced in this investigation with studies of CD4⁺ T cell functions such as cytokine-producing capacity.

	FOOTNOTES

 
¹ Supported by grants awarded to B.D.W. by the Natural Sciences and Engineering Research Council of Canada and by the Nutricia Research Foundation (Holland).

³ Abbreviations used: PEM, protein-energy malnutrition; SRBC, sheep red blood cells; T, thymus.

Manuscript received November 29, 2000. Initial review completed January 11, 2001. Revision accepted March 26, 2001.

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