Balanced T_H1 and T_H2 immunopotentiating effects of silicates partly containing nanoparticles present in calcined serpentine

Figures & data

Figure 1. TEM analyses. Analyses of CS samples were performed using a JEOL 2100F transmission electron microscope. (a) Determination of particle size. (b) Size distribution of NP present in the CS sample. Representative analyses are shown.

Figure 2. XRD analysis of CS samples. Plotting of the 2-theta values vs signal intensity (in Arbitrary Units [AU]) allowed for identification of the indicated mineral constituents. P = Phillipsite, A = Actinolyte, C = Clinoptilolite, M = Muscovite, D = Dolomite, E = Epidote, Z = Zoisite.

Table 1. Characterization of CS.

ICP-MS
Elements	% in CS	Board-recommended value	Content in 200 μg CS
Na	0.026	2.40 g/day	0.52 μg/day
Mg	16.60	0.35 g/day	33.20 μg/day
K	0.40	3.50 g/day	8.00 μg/day
Fe	9.84	0.04 g/day	19.68 μg/day
P	7.78	4.00 g/day	15.50 μg/day
S	14.63	Not specified	29.26 μg/day
Atomic absorption spectroscopy
Metal	Content in CS	WHO limit (25 g mouse)	Presence in daily 200 μg CS dose
Lead	168.93 mg/kg	1.20 μg/day	3.30 ng/day
Mercury	1.10 mg/kg	0.24 μg/day	0.02 ng/day
Arsenic	Not detected	0.60 μg/day	—
Cadmium	Not detected	—	—
CHNS analysis
Elements	Weight in CS (mg)	Content (%)	C:N ratio
Nitrogen	5.1500	0.123	4.944
Carbon		0.609
Sulfur		0.190
Hydrogen		0.797

Specific analyses were performed as outlined in the Methods.

Table 2. Humoral immune response-related endpoints.

CS groups	Total IgG	IgG1	IgG2a	IgG2b	IgG3
Bleed I
Control	0.91 ± 0.04	0.20 ± 0.01	0.18 ± 0.01	0.21 ± 0.01	0.21 ± 0.01
50 μg/kg	0.94 ± 0.04	0.20 ± 0.01	0.18 ± 0.01	0.21 ± 0.01	0.21 ± 0.01
75 μg/kg	1.08 ± 0.07*	0.22 ± 0.01*	0.19 ± 0.01	0.21 ± 0.01	0.21 ± 0.01
100 μg/kg	1.09 ± 0.04*	0.22 ± 0.01*	0.19 ± 0.01	0.21 ± 0.01	0.22 ± 0.01
200 μg/kg	1.05 ± 0.04	0.21 ± 0.01	0.18 ± 0.01	0.21 ± 0.01	0.21 ± 0.01
Bleed II
Control	1.40 ± 0.02	0.20 ± 0.02	0.17 ± 0.01	0.19 ± 0.01	0.33 ± 0.01
50 μg/kg	1.41 ± 0.02	0.31 ± 0.01**	0.32 ± 0.01**	0.33 ± 0.01**	0.32 ± 0.01
75 μg/kg	1.74 ± 0.02**	0.31 ± 0.01**	0.36 ± 0.02**	0.33 ± 0.01**	0.36 ± 0.01
100 μg/kg	1.83 ± 0.03**	0.32 ± 0.01**	0.36 ± 0.01**	0.33 ± 0.01**	0.33 ± 0.02
200 μg/kg	1.77 ± 0.02**	0.32 ± 0.01**	0.35 ± 0.01**	0.32 ± 0.01**	0.32 ± 0.01
Bleed III
Control	2.09 ± 0.04	0.69 ± 0.02	0.27 ± 0.01	0.29 ± 0.01	0.45 ± 0.01
50 μg/kg	2.25 ± 0.02**	0.72 ± 0.01**	0.79 ± 0.01**	0.77 ± 0.01**	0.71 ± 0.01**
75 μg/kg	2.32 ± 0.02**	0.78 ± 0.01**	0.80 ± 0.01**	0.84 ± 0.01**	0.67 ± 0.01**
100 μg/kg	2.36 ± 0.02**	0.79 ± 0.01**	0.80 ± 0.01**	0.93 ± 0.01**	0.54 ± 0.01**
200 μg/kg	2.29 ± 0.02**	0.77 ± 0.02**	0.57 ± 0.01**	0.73 ± 0.01**	0.50 ± 0.02**
Bleed IV
Control	1.88 ± 0.03	0.69 ± 0.01	0.26 ± 0.01	0.28 ± 0.01	0.43 ± 0.01
50 μg/kg	2.22 ± 0.02**	0.72 ± 0.01	0.68 ± 0.01**	0.75 ± 0.02**	0.68 ± 0.01**
75 μg/kg	2.31 ± 0.02**	0.77 ± 0.01**	0.78 ± 0.01**	0.82 ± 0.01**	0.66 ± 0.01**
100 μg/kg	2.21 ± 0.03**	0.78 ± 0.01**	0.78 ± 0.01**	0.91 ± 0.01**	0.52 ± 0.01**
200 μg/kg	2.17 ± 0.03**	0.76 ± 0.02*	0.56 ± 0.01**	0.72 ± 0.01**	0.49 ± 0.02**

Values shown are means ± SE (n = 5).

* p < 0.05.

** p < 0.01 vs respective control.

Table 3. Effects of CS on cell-mediated and innate immunity-related endpoints.

Endpoints	Control	50 μg CS/kg	75 μg CS/kg	100 μg CS/kg	200 μg CS/kg
CMI response
DTH response	15.60 ± 2.12	18.52 ± 5.78	29.42 ± 0.03*	37.52 ± 1.05**	35.94 ± 1.81**
Lymphoproliferation Unstimulated
OVA-stimulated	0.395 ± 0.012	0.417 ± 0.006	0.421 ± 0.006	0.423 ± 0.004	0.422 ± 0.007
ConA-stimulated	0.617 ± 0.006	0.653 ± 0.005*	0.657 ± 0.006*	0.668 ± 0.011**	0.644 ± 0.008
Innate response	0.817 ± 0.026	0.981 ± 0.008**	1.022 ± 0.006**	1.321 ± 0.011**	1.125 ± 0.012**
PEM NO induction:
Unstimulated	9.23 ± 0.38	10.02 ± 0.34	10.22 ± 0.171	10.52 ± 0.15	9.99 ± 0.33
LPS stimulated	17.23 ± 0.36	19.05 ± 0.73	19.25 ± 0.606	19.60 ± 0.52	19.13 ± 0.46
PEM Phagocytic rate (%)	18.30 ± 2.02	27.30 ± 1.45**	33.60 ± 1.76**	38.30 ± 2.18**	33.00 ± 2.08**
PEM Phagocytic index	1.03 ± 0.02	1.08 ± 0.01	1.10 ± 0.01*	1.15 ± 0.01**	1.12 ± 0.02*

DTH is expressed as footpad thickness (in mm) 24 h after challenge with GRBC. Lymphoproliferation is expressed as absorbance values for non-, OVA- or ConA-treated spleen cells in culture.

With peritoneal macrophages (PEM), NO formation (spontaneous and LPS-induced) is expressed as absorbance values. Phagocytic responses are expressed as phagocytic rate and index.

All data shown are means ± SE (n = 5).

* p < 0.05.

** p < 0.01 vs control.

Figure 3. Ex vivo cytokine formation by mouse splenocytes. Splenocytes were isolated from mice in the different test groups and then the cells were plated (2 × 10⁵ cells/well) and then stimulated with ConA (2 μg/ml) or OVA (5 μg/ml) for 72 h. Culture supernatants were then collected and analyzed for the indicated T_H1 and T_H2 cytokines by ELISA. Values shown are mean ± SE (n = 5). *p< 0.01, #p < 0.05 compared to respective control.

Figure 4. Ex vivo TNFα and IL-1β formation by mouse splenocytes. Splenocytes were isolated from mice in the different test groups and then the cells were plated (2 × 10⁵ cells/well) and then stimulated with ConA (2 μg/ml) or OVA (5 μg/ml) for 72 h. Culture supernatants were then collected and analyzed for the indicated pro-inflammatory cytokines by ELISA. Values shown are mean ± SE (n = 5).

Figure 5. Absorption of CS in rat intestine everted gut sac. Intestinal sacs were incubated in solutions of the indicated CS sample (200 μg/ml) or buffer only for 45 min. Values are expressed as mean ± SE (n = 5). **p < 0.01 compared with control.

Figure 6. Flow cytometric analysis of peritoneal macrophage TLR-2 and TLR-4 expression. Isolated cells were labeled with AF647-labeled anti-TLR-2 and PE-anti-TLR-4. Representative dot-plots are shown. (a) Control, (b) 50 μg CS/kg, (c) 75 μg CS/kg, (d) 100 μg CS/kg and (e) 200 μg CS/kg dose.

Figure 7. Flow cytometric analysis of peritoneal macrophage co-stimulatory CD80 and CD86 expression. Isolated cells were labeled using FITC anti-CD-80 and PE-anti-CD86. Representative dot-plots are shown. (a) Control, (b) 50 μg CS/kg, (c) 75 μg CS/kg, (d) 100 μg CS/kg, and (e) 200 μg CS/kg dose.

Figure 8. Histopathologic damage due to CS. The mice were daily gavaged with 200 μg CS/kg or vehicle only for 10 days. At 24 h after the final dosing, the host liver, kidney, and spleen were isolated and prepared for histopathology. Representative sections are shown; Magnification = 10–40×. (I) Control mouse kidney, (II) Control mouse liver, (III) Control mouse spleen, (IV) CS mouse kidney, (V) CS mouse liver and (VI) CS mouse spleen.