Decrease of Hyaluronidase Activity and Suppression of Mouse CD4+ T Lymphocyte Activation by Tomato Juice Saponin Esculeoside B, and Its Sapogenol Esculeogenin B

(1) Background: A naturally occurring glycoside, esculeoside B (EsB), has been identified as a major component in juice or canned tomato. We reported how EsB ameliorated mice experimental atopic dermatitis by a decrease in serum IgE levels. However, the underlying immunologic molecular mechanisms are unknown. (2) Methods: The present study tested the effects of EsB on hyaluronidase activity and CD4+ T lymphocyte activation using concanavalin A (ConA)-blast mouse splenocyte primary culture. (3) Results: We found that EsB and its sapogenol esculeogenin B (Esg-B) decreased hyaluronidase activity by a modified Morgan–Elson method. We demonstrated that EsB/Esg-B dose-dependently suppressed T-lymphoproliferation using CFSE-labeled flow-cytometry and water-soluble tetrazolium (WST) assay. Using ELISA and q-PCR methods, EsB/Esg-B suppressed the cytokine secretion and mRNA expression of Th2-relevant IL-4 and Th1-relevant IFN-γ. Moreover, both EsB/Esg-B showed a reduction in IL-10 secretion, but only Esg-B decreased IL-2 secretion. (4) Conclusions: Our study is the first to demonstrate how EsB/Esg-B inhibit hyaluronidase activity and reduce CD4+ T-lymphocyte activation via a reduction in Th2-lymphocyte activity by modulation of Th2/Th1/Treg subunits differentiation.


Introduction
Atopic dermatitis (AD) is a chronic inflammatory disorder predominantly mediated by CD4+ T helper cells [1][2][3][4]. First, an overexpression of Th2 cell cytokines was observed in acute and chronic lesions of AD. The Th2 response is characterized by skewed cytokine production including IL-4 and IL-5, and associated downstream GATA binding protein 3 (GATA3), which in turn drive eosinophilic inflammation and immunoglobulin class switching to IgE in B cells and subsequent release of inflammatory mediators [5]. Second, it has been reported that chronic lesions also express moderate levels of the Th1 cell cytokine IFN-γ; thus, the T cell response present in AD is not only Th2-polarized but may lead to heterogeneous cytokine production involving Th1 cell cytokines [6]. Third, it has been reported that children with AD had higher Th2, memory Regulatory T (Treg) cell numbers compared to healthy children. While higher memory Treg cells represent chronic inflammation, naive cells have no memory of sensitization to specific antigens [7][8][9]. The major population of CD4+ Treg cells was found to be characterized by high expression of IL-2 receptor alpha chain (CD25) and transcription factor Foxp3 (forkhead box P3), which are important for the development and suppressive function of Treg cells.
Moreover, it is well know that hyaluronidase, an enzyme for hyaluronic acid (HA) degradation, is related to inflammation and allergic responses [10]. It has been reported that, during inflammation and tissue damage, hyaluronidase cleaves polysaccharide HA in low molecular weight HA fragments [11], which induce pro-inflammatory immune responses [12].
We identified esculeosides B (EsB), which contains four times the amount of lycopene content, as a major component in juice or canned tomato [13,14]. Concerning its chemical structure, EsB is a rare and naturally occurring compound which is a solanocapsine-type glycoside. Our previous study demonstrated how EsB ameliorated mice experimental atopic dermatitis by a decrease in serum IgE levels [15]. However, the underlying antiinflammatory mechanisms are unknown.
Therefore, the present study tested the effects of EsB on hyaluronidase activity and mouse CD4+ T lymphocyte activation. The results demonstrated how EsB and its sapogenol esculeogenin B (Esg-B) inhibited hyaluronidase activity, and suppressed immune activation in mouse CD4+ T cells following in vitro stimulation with concanavalin A (ConA).

Preparation of EsB and Esg-B
EsB was extracted [14]; 900 g of commercially processed tomato juice (Solanum lycopersicum L.) was centrifuged. The filtrate passed through a highly porous polystyrene gel (Diaion ® HP-20, Mitsubishi Chemical, Tokyo, Japan). The resulting methanol residue was then subjected to dextran gel column (Sephadex ® LH-20, Sigma-Aldrich, St. Louis, MO, USA) chromatography and afterwards eluted with 90% methanol. The 90% methanol eluates lacked esculeoside A (EsA) but contained EsB on TLC with CHCl 3 -MeOH-H 2 O (6:4:1). Next, the obtained tomato saponin was identified by the NMR spectrum. The average yield of EsB from commercially produced tomato juice was calculated to be about 0.041%. After a solution of EsB (350 mg) in 2 N HCl was refluxed for 1 h, its reaction mixture was neutralized with 2 N KOH and water was added; next it was passed through Diaion HP-20 and washed with water, then eluted with methanol to give a sapogenol. Crude sapogenol was purified on silica gel column chromatography with CHCl 3 -MeOH-H 2 O (9:1:0.1) to afford the sapogenol Esg-B (25 mg). The chemical structure of EsB is shown in Figure 1A.

Hyaluronidase Inhibitory Assay
The inhibitory activity on hyaluronidase was measured according to a modified Morgan-Elson method as described previously [16,17]. Briefly, samples prepared in 0.1 M acetate buffer (pH 4) and hyaluronidase (type IV-S: From bovine testes, Sigma, St. Louis, MO, USA) in buffer with a final concentration of 5 mg/mL were incubated at 37 • C. Then, compound 48/80 (Sigma) in buffer with a final concentration of 0.5 mg/mL was added and incubated. After hyaluronic acid sodium salt (from rooster comb, Wako, Osaka, Japan) in buffer with a final concentration of 0.4 mg/mL had been included, the mixture was incubated. p-Dimethylaminobenzaldehyde (Wako) acetate solution was then added and incubated. Then the absorbance was determined at 585 nm, and the enzyme inhibitory activity (%) was quantified. EsB or Esg-B as a stock solution (2.8 mg/mL) was prepared with 0.7% DMSO and diluted with 0.1 M acetate buffer. Disodium chromoglycate (Wako) and suplatast tosylate (TCI, Tokyo, Japan) were prepared with water as a stock solution of 10 mg/mL. yield of EsB from commercially produced tomato juice was calculated to be about 0.041% After a solution of EsB (350 mg) in 2 N HCl was refluxed for 1 h, its reaction mixture was neutralized with 2 N KOH and water was added; next it was passed through Diaion HP 20 and washed with water, then eluted with methanol to give a sapogenol. Crude sap ogenol was purified on silica gel column chromatography with CHCl3-MeOH-H2O (9:1:0.1) to afford the sapogenol Esg-B (25 mg). The chemical structure of EsB is shown in

A.
Esculeogenin B (Esg-B), 22R, 23S Esculeoside B (EsB) Glc Glc xyl Glc Gal yield of EsB from commercially produced tomato juice was calculated to be about 0.041% After a solution of EsB (350 mg) in 2 N HCl was refluxed for 1 h, its reaction mixture wa neutralized with 2 N KOH and water was added; next it was passed through Diaion HP 20 and washed with water, then eluted with methanol to give a sapogenol. Crude sap ogenol was purified on silica gel column chromatography with CHCl3-MeOH-H2O (9:1:0.1) to afford the sapogenol Esg-B (25 mg). The chemical structure of EsB is shown in Figure 1A.

Animals and Splenocyte Isolation
The study was submitted to and approved by the Ethics Committee of Sojo University (2019-P-025, 2020-P-025). All experiments were conducted in strict accordance with the Guidelines of the Japanese Pharmacological Society for the Care and Use of Laboratory Animals. Female BALB/c mice, 6~9 weeks old, were obtained from Japan SLC (Hamamatsu, Japan). The animals were housed under conditions of controlled room temperature (24.5-25.0 • C), 60 ± 10% humidity, and a 12/12 h light/dark cycle. Food pellets and water were provided ad libitum. Mice were sacrificed by exposure to isoflurane; the spleen was dissected out and immediately immersed in PBS, then minced and passed through a 70 µm cell strainer. After treatment with RBC lysis buffer to deplete red blood cells, splenocytes were suspended in an RPMI 1640 medium (Wako, Osaka, Japan) supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin (Invitrogen, Waltham, MA, USA) at a density of 3 × 10 6 cells/mL in 96-well or 24-well plates, and cultured under a humidified atmosphere of 5% CO 2 at 37 • C before being subjected to various treatments.
Splenocyte proliferation was also analyzed by WST (water-soluble tetrazolium) assay using a Cell Count Kit-8 (CCK-8) (Dojindo, Kumamoto, Japan). After seeding, splenocytes were pretreated with EsB or Esg-B, stimulated with ConA, and cultured for 3 days. Next, the cells were incubated with CCK-8 for 5 h, and the absorbance was recorded using a micro-plate reader at 450 nm. The proliferation rate was compared with that of the untreated control group.

Quantitative Polymerase Chain Reaction (q-PCR) Assay
Quantification of mRNA expression in splenocytes was performed using RT-PCR by subjecting the cDNA obtained from the following preparation methods to PCR amplification using a StepOnePlus™ Real-Time PCR System (Life Technologies, Waltham, MA, USA).
Total RNA from mouse splenocytes was isolated from homogenates using an RNeasy Mini Kit (Qiagen, Düsseldorf, Germany). The obtained mRNA was quantified by measuring the absorbance at 260 nm, and its quality was determined by measuring the 260/280 ratio. cDNA was synthesized using a PrimeScriptTM 1st strand cDNA Synthesis kit (Takara Bio, Kusatsu, Japan). In brief, 1.0 µg of total RNA from each sample was added to a mixture of 1.0 µL of random 6 mers (50 µM), 1.0 µL of dNTP mix (10 mM), and 8.0 µL of RNase-free dH 2 O, and then held at 65 • C for 5 min and cooled to 4 • C using a thermocycler (Eppendorf, Hamburg, Germany). Next, the denatured mixture was added with 4.0 µL of 5× PrimeScript buffer, 0.5 µL of RNase inhibitor, 1.0 µL of PrimeScript RTase, and 4.5 µL of RNase-free dH2O, and then held at 50 • C for 45 min, heated to 95 • C for 5 min and cooled to 4 • C. Finally, the 20-µL PCR reaction mixture contained 1.2 µL of 10 M forward primers and 1.2 µL of 10 M reverse primers (300 nM final concentration of each primer), 10 µL of PowerUpTM SYBR ® Green Master mix (2×), 5.6 µL of nuclease-free water, and 2.0 µL of the obtained cDNA. Thermal cycling conditions were applied as follows: 50 • C for 2 min, 95 • C for 2 min, followed by 40 cycles of 15 s at 95 • C and 1 min at 60 • C. The samples were matched to a standard curve generated by amplifying serially diluted products using the same real-time PCR conditions. The data are presented as the fold change (2 −∆∆Ct ) in gene expression levels and normalized to the mRNA expression levels of an endogenous reference gene, GAPDH (glyceraldehyde 3-phosphate dehydrogenase), then shown relative to that of the control group [19].
The primer sequences of the target genes, selected from PubMed and other databases, were as follows (Table 1): Table 1. Quantitative polymerase chain reaction primers for GAPDH, IL-4, IFN-γ, mRNA.

Forward
Reverse The data are presented as the mean ± SEM of at least three independent experiments and analyzed by Prism 8 (GraphPad Software, San Diego, CA, USA). Student's T-test was used to determine statistical significance between two groups, and one-way ANOVA with Tukey's multiple comparison test was used for multiple groups. p-values < 0.05 were considered to be significantly different.

Hyaluronidase Inhibition by EsB/Esg-B
Firstly, the effects of EsB/Esg-B, chromoglycate, and suplatast on hyaluronidase activity were determined in vitro. As shown in Figure 1B, EsB/Esg-B inhibited hyaluronidase activity in a concentration-dependent manner, and IC50 value was about 200 µM. Chromoglycate induced concentration-dependent inhibition on hyaluronidase with about 10 µM of IC50. Suplatast did not inhibit hyaluronidase activity.

Cytotoxicity of EsB/Esg-B in Splenocytes
Our previous study demonstrated that the oral administration of EsB at 10 mg/kg or fresh tomato fruits saponin EsA at 10-100 mg/kg ameliorated experimental dermatitis in mice [15]. It has been reported that esculeogenin A (Esg-A), a sapogenol of EsA, at 3-100 µM inhibited the accumulation of cholesterol ester in human monocyte-derived macrophages [20]. Considering these in vivo and in vitro effects, various concentrations of EsB and Esg-B were tested for cytotoxicity to splenocytes as shown in Figure 2. When the live cell rate of the control was normalized to 1.0, the ratios of live splenocytes were 1 The data are presented as the mean ± SEM of at least three independent experime and analyzed by Prism 8 (GraphPad Software, San Diego, CA, USA). Student's T-test w used to determine statistical significance between two groups, and one-way ANOVA w Tukey's multiple comparison test was used for multiple groups. p-values < 0.05 were co sidered to be significantly different.

Hyaluronidase Inhibition by EsB/Esg-B
Firstly, the effects of EsB/Esg-B, chromoglycate, and suplatast on hyaluronidase tivity were determined in vitro. As shown in Figure 1B, EsB/Esg-B inhibited hyaluro dase activity in a concentration-dependent manner, and IC50 value was about 200 μ Chromoglycate induced concentration-dependent inhibition on hyaluronidase with abo 10 μM of IC50. Suplatast did not inhibit hyaluronidase activity.

Cytotoxicity of EsB/Esg-B in Splenocytes
Our previous study demonstrated that the oral administration of EsB at 10 mg/kg fresh tomato fruits saponin EsA at 10-100 mg/kg ameliorated experimental dermatitis mice [15]. It has been reported that esculeogenin A (Esg-A), a sapogenol of EsA, at 3-1 μM inhibited the accumulation of cholesterol ester in human monocyte-derived mac phages [20]. Considering these in vivo and in vitro effects, various concentrations of E and Esg-B were tested for cytotoxicity to splenocytes as shown in Figure 2. When the l cell rate of the control was normalized to 1.0, the ratios of live splenocytes were 1.01, 0.

Suppression of T Lymphocyte Proliferative Response by EsB/Esg-B
Effects of EsB/Esg-B on T lymphocyte proliferative response were tested using vitro ConA-stimulated T cell proliferation (Figure 3). T lymphocyte proliferative effect the culture with EsB/Esg-B was compared to that without EsB/Esg-B (ConA alone).

Suppression of T Lymphocyte Proliferative Response by EsB/Esg-B
Effects of EsB/Esg-B on T lymphocyte proliferative response were tested using in vitro ConA-stimulated T cell proliferation (Figure 3). T lymphocyte proliferative effect in the culture with EsB/Esg-B was compared to that without EsB/Esg-B (ConA alone). As shown in Figure 3A,B, using the WST assay, addition of EsB/Esg-B to the culture system resulted in a concentration-dependent suppression of the proliferation response. Esg-B at about 5 µM inhibited the elevated proliferation ratio by 50%, whereas EsB at about 120 µM was required to show the same inhibition. In Figure 4, further assays using CD3-PE+/CFSE+ fluorescence demonstrated whether or not this dose-dependent suppression by EsB/Esg-B was related to T cell division. Daughter T cells ( Figure 4A, R2), derived from responder splenocytes following ConA stimulation, were differentiated from undivided T cells (R1) and identified by the intensity of CFSE staining ( Figure 4A,B) using flow cytometry. The addition of EsB/Esg-B to CFSE-labeled cells stimulated with ConA showed a tendency to suppress T lymphocyte division ( Figure 4B,C). Esg-B at about 3 μM decreased dividing T cells by 50%, whereas about 100 μM EsA was needed to show the same reduction. In Figure 4, further assays using CD3-PE+/CFSE+ fluorescence demonstrated whether or not this dose-dependent suppression by EsB/Esg-B was related to T cell division. Daughter T cells ( Figure 4A, R2), derived from responder splenocytes following ConA stimulation, were differentiated from undivided T cells (R1) and identified by the intensity of CFSE staining ( Figure 4A,B) using flow cytometry. The addition of EsB/Esg-B to CFSE-labeled cells stimulated with ConA showed a tendency to suppress T lymphocyte division ( Figure 4B,C). Esg-B at about 3 µM decreased dividing T cells by 50%, whereas about 100 µM EsA was needed to show the same reduction.

EsB/Esg-B Modulation of ConA Blast-Induced Cytokine Level
During T cell activation, cytokines are produced to modulate the differentiation and subsequent specialization of T cells; thus, we examined the effects of EsB/Esg-B on ConA blast-induced cytokine extracellular secretion. EsB/Esg-B decreased the production of Th2 cytokine IL-4 and the Th1 cytokine IFN-γ in a concentration-dependent manner, as shown in Figure 5A. In detail, Esg-B at about 2 μM inhibited the elevated IL-4 and IFN-γ

EsB/Esg-B Modulation of ConA Blast-Induced Cytokine Level
During T cell activation, cytokines are produced to modulate the differentiation and subsequent specialization of T cells; thus, we examined the effects of EsB/Esg-B on ConA blast-induced cytokine extracellular secretion. EsB/Esg-B decreased the production of Th2 cytokine IL-4 and the Th1 cytokine IFN-γ in a concentration-dependent manner, as shown in Figure 5A. In detail, Esg-B at about 2 µM inhibited the elevated IL-4 and IFN-γ production by 50%, whereas EsB at about 30 µM and 20 µM was required to show the same inhibition, respectively, indicating the similar inhibitory effect of EsB/Esg-B on Th2 and Th1 cytokine production. Further, the inhibitory effect of EsB/Esg-B on Th2/Th1 cytokine production is greater than for IL-2 production. Moreover, EsB/Esg-B also decreased production of the Treg cytokine IL-10, which are required for Treg cell maintenance. In detail, Esg-B at about 1 µM inhibited the elevated IL-10 production by 50%, whereas EsB at about 10 µM was required to show the same inhibition. production by 50%, whereas EsB at about 30 μM and 20 μM was required to show the same inhibition, respectively, indicating the similar inhibitory effect of EsB/Esg-B on Th2 and Th1 cytokine production. Further, the inhibitory effect of EsB/Esg-B on Th2/Th1 cytokine production is greater than for IL-2 production. Moreover, EsB/Esg-B also decreased production of the Treg cytokine IL-10, which are required for Treg cell maintenance. In detail, Esg-B at about 1 μM inhibited the elevated IL-10 production by 50%, whereas EsB at about 10 μM was required to show the same inhibition.
We also examined the effect of EsB/Esg-B on ConA blast-stimulated cytokine mRNA expression. EsB/Esg-B decreased mRNA expression levels of IL-4 and IFN-γ in a concentration-dependent manner, as shown in Figure 5B. In detail, Esg-B at both 10 μM inhibited the elevated IL-4 and IFN-γ gene expression levels by 50%, whereas EsB at both 100 μM were required to show the same inhibition, respectively, also indicating the similar inhibitory effect of EsB/Esg-B on Th2 and Th1 gene expression level. We also examined the effect of EsB/Esg-B on ConA blast-stimulated cytokine mRNA expression. EsB/Esg-B decreased mRNA expression levels of IL-4 and IFN-γ in a concentration-dependent manner, as shown in Figure 5B. In detail, Esg-B at both 10 µM inhibited the elevated IL-4 and IFN-γ gene expression levels by 50%, whereas EsB at both 100 µM were required to show the same inhibition, respectively, also indicating the similar inhibitory effect of EsB/Esg-B on Th2 and Th1 gene expression level.

Discussion
In this in vitro study, it showed that the saponin EsB and its sapogenol Esg-B from tomato juice alleviate ConA-blast T lymphocyte activity by modulation of Th1/Th2/Tregassociated cytokines signaling.
Our previous study showed that oral administration of EsB, a solanocapsine-type glycoside and a major component in tomato juice, ameliorated experimental dermatitis in mice through decreases in IgE and ConA-mitogenic action, and a decline in IL-4 secretion [14,15]. However, the underlying immunologic molecular mechanisms are unknown. Hence, the present study investigated the effects of EsB/Esg-B on ConA-blast mouse splenocytes, and analyzed the immune mechanism in relation to CD4+ T lymphocytes. We pretested the EsB/Esg-B cytotoxic effects in mouse primary splenocytes, and found that cytotocity was lower at the concentrations of EsB < 150 µM and Esg-B < 30 µM (Figure 2). Based on these concentrations, we first tested the effects of EsB/Esg-B on T lymphoproliferative action induced by ConA, a selective T cell mitogen. Using WST assay, EsB/Esg-B suppressed splenocyte proliferation (Figure 3), and showed profound suppression of CD3+ T lymphocyte division by flow cytometry (Figure 4). Both assays showed that EsB/Esg-B decreased T lymphocyte proliferation; however, the latter may suggest their potential effect on T lymphocytes. The present T lymphoproliferative decline by EsB is in line with our previous in vivo study using EsB [15].
We subsequently tested EsB/Esg-B for their Th2/Th1 cytokine production-modulatory potential, since in the atopy patch test it was observed that T cells in the skin display an initial Th2 polarization, with increasing populations of Th1 cells in patients with chronic AD [21][22][23]. The high proportion of Th2-polarized T cells appears to be a key factor in patients with allergic inflammation [24]. The present study used the ConA-stimulated lymphoproliferative system, and it was found that EsB/Esg-B suppressed both Th2/Th1 cytokine production and intracellular gene expression levels ( Figure 5). Moreover, EsB/Esg-B inhibitory degrees on Th2-mediated cytokine production and gene expression were similar to those on Th1-mediated responses. Recently, the clinical efficacy of the IL-4 receptor antagonist dupilumab was demonstrated, in addition to a decline in Th1 and Th2 cell numbers [25,26], supporting evidence for the importance of the Th2/Th1 immune pathway in AD. Thus, we highlight that EsB/Esg-B can suppress Th2/Th1 dominant inflammatory reaction.
In addition to Th2 and Th1 effector cells, CD4+ Th cells are controlled by Treg cells, which regulate inflammatory responses and restore immune homeostasis. We examined the Treg-modulatory potential of EsB/Esg-B, and demonstrated that EsB/Esg-B suppressed Treg cytokine IL-10 production ( Figure 5). This indicated that EsB/Esg-A alleviated the suppressive action of IL-10-producing Treg cells in mouse splenocytes in response to ConA mitogenic stimuli. At the same time, Esg-B modulation in CD4+ Th cells is characterized by a reduction in IL-2 signaling, including IL-2 cytokine secretion ( Figure 5). Since IL-2 has been reported as an important activator of Treg suppressive activity in vitro and in vivo [27][28][29][30], and the present data suggests that during suppressed IL-2 signaling by Esg-B, the concomitant suppression of Treg and/or the other immune cells may still counteract ConA-induced in vitro activation of cellular immunity. On the other hand, EsB did not affect IL-2 production, a point that needs to be examined further.
Moreover, this study also indicates that the inhibitory effect of EsB/Esg-B on mammalian hyaluronidase activity is weaker than that of chromoglycate, an anti-allergic agent, known as a mast cell stabilizer, which inhibits the release of mediators, such as histamine and leukotrienes, and is stronger than that of suplatast, another anti-allergic agent, which is able to alleviate cutaneous symptoms of atopic dermatitis and nasal symptoms of allergic rhinitis by inhibiting the production of Th2 cytokine and immunoglobulin E [31,32], and which also suppresses histamine release and H1 receptor expression [33]. Thus, further research in chemical mediator release is warranted for molecular targets of anti-allergic therapeutic effects of EsB/Esg-B. Moreover, their inhibitory activity on the hyaluronidase activity might be not only in managing atopic dermatitis, but also in approaching intestinal diseases, exactly through hyaluronic acid degradation in the intestinal bio-film.
Finally, in a comparison of EsB-and Esg-B-suppressive potential in Th2/Th1/Treg activation, the suppression by Esg-B is greater than that by EsB, suggesting that the steroidal alkaloid Esg-B moiety may be mainly responsible for the suppressive effect on CD4+ T cell activity at least in vitro. The main effect of Esg-B should be consistent with our previous report, in which the final metabolites were eliminated as androsterone analogues in the urine of men orally administered ripe cherry tomato fruit [34].

Conclusions
Taken together, these data indicate that tomato juice saponin EsB and its sapogenol Esg-B are capable of down-regulating in vitro ConA-mitogenic CD4+ T lymphocyte activation via modulating the differentiation of Th2, Th1, and Treg subsets, thereby contributing to the EsB-mediated alleviation of experimental dermatitis in mice. Next steps include investigation of their downstream stimuli and the signaling pathways affecting the Th2/Th1/Treg cells. This section is not mandatory but can be added to the manuscript if the discussion is unusually long or complex.  Institutional Review Board Statement: The study was submitted to and approved by the Ethics Committee of Sojo University (2019-P-025, 2020-P-025). All experiments were conducted in strict accordance with the Guidelines of the Japanese Pharmacological Society for the Care and Use of Laboratory Animals.