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Volume 30, Issue 3, Pages 350-357 (March 2014) Gestational vitamin A deficiency reduces the intestinal immune response by decreasing the number of immune cells in rat offspring Xia Liu, M.D., Yingying Li, M.D., Yuting Wang, M.D., Qinghong Wang, Ph.D., Xin Li, M.D., Yang Bi, Ph.D., Lan Liu, M.D., Xiaoping Wei, M.D., Tingyu Li, M.D., Jie Chen, Ph.D. Nutrition Volume 30, Issue 3, Pages 350-357 (March 2014) DOI: 10.1016/j.nut.2013.09.008 Copyright © 2014 Elsevier Inc. Terms and Conditions

Fig. 1 Schematic diagram showing the three offspring groups: VAN, VAD, and VAD-N. The VAN pups were delivered and nursed by maternal VAN rats and then fed a VAN diet for 3 wk after weaning. The VAD pups were birthed and nursed by maternal VAD rats and fed a VAD diet for 3 wk after weaning. The VAD-N pups were delivered from maternal VAD rats and then nursed by maternal VAN rats and fed with a VAN diet until they were 6 wk old. VAD, vitamin A deficiency; VAD-N, VAD during gestation and fed the milk of VAN rats after birth; VAN, vitamin A normal. Nutrition 2014 30, 350-357DOI: (10.1016/j.nut.2013.09.008) Copyright © 2014 Elsevier Inc. Terms and Conditions

Fig. 2 Effects of VAD on the levels of serum retinol, IgA secretion, and the histopathologic changes of intestinal mucosa in 6-wk-old pups. (A) The changes in the serum retinol levels (μmol/L) among the VAN, VAD, and VAD-N groups after the presence or absence of Escherichia coli LPS challenge as assessed by HPLC (n = 15). (B) The VA main effect, independent of LPS treatment, as shown by the serum retinol levels (μmol/L) among the combined VAN, VAD, and VAD-N groups. (C) The levels of IgA in the intestinal stool among the VAN, VAD, and VAD-N groups with or without LPS treatment (n = 6). (D) Histologic examination of intestinal mucosa staining with hematoxylin and eosin (n = 3). “Interaction” indicates an effect of LPS treatment versus null treatment. * P < 0.05; † P < 0.01; ‡ P < 0.001; n.s. = not significant in post hoc tests. HPLC, high performance liquid chromatography; IgA, immunoglobulin A; LPS, lipopolysaccharide; VAD, vitamin A deficiency; VAD-N, VAD during gestation and fed the milk of VAN rats after birth; VAN, vitamin A normal. Nutrition 2014 30, 350-357DOI: (10.1016/j.nut.2013.09.008) Copyright © 2014 Elsevier Inc. Terms and Conditions

Fig. 3 The percentage of (A) CD4+CD25+ T cells and (C) CD4+CD8+ double-positive T lymphocytes in the spleen of VAN, VAD, and VAD-N groups with or without LPS challenge assessed with the use of flow cytometry (n = 8). (B) The LPS main effect, independent of VA treatment, in the percentage of CD4+CD25+ T cells in the spleen. “Interaction” indicates an effect of LPS treatment versus null treatment. *P < 0.05, †P < 0.01; ‡P < 0.001; n.s.= not significant in post hoc tests. LPS, lipopolysaccharide; VAD, vitamin A deficiency; VAD-N, VAD during gestation and fed the milk of VAN rats after birth; VAN, vitamin A normal. Nutrition 2014 30, 350-357DOI: (10.1016/j.nut.2013.09.008) Copyright © 2014 Elsevier Inc. Terms and Conditions

Fig. 4 Increase in the number of B lymphocytes in the MLN as a result of (A) gestational VAD (n = 8 to 10) and (B) the VA significant main effect, independent of LPS treatment, on the percentage of B cells in the MLN among the three combined VAN, VAD, and VAD-N groups. “Interaction” indicates an effect of LPS treatment versus null treatment. ‡P < 0.001; n.s.= not significant in post hoc tests. LPS, lipopolysaccharide; MLN, mesenteric lymph node; VAD, vitamin A deficiency; VAD-N, VAD during gestation and fed the milk of VAN rats after birth; VAN, vitamin A normal. Nutrition 2014 30, 350-357DOI: (10.1016/j.nut.2013.09.008) Copyright © 2014 Elsevier Inc. Terms and Conditions

Fig. 5 Suppression of (A) CD11 C+ dendritic cells and (C) CD4+CD25+ T cells in PPs by gestational VAD in the presence or absence of LPS treatment (n = 5 to 6). The VA significant main effect, independent of LPS treatment, on the percentage of (B) CD11 C+ dendritic cells and (D) CD4+CD25+ T cells in PPs among the three combined VAN, VAD, and VAD-N groups. “Interaction” indicates an effect of LPS treatment versus null treatment. ‡P < 0.001; n.s.= not significant in post hoc tests. LPS, lipopolysaccharide; PPs, Peyer patches; VAD, vitamin A deficiency; VAD-N, VAD during gestation and fed the milk of VAN rats after birth; VAN, vitamin A normal. Nutrition 2014 30, 350-357DOI: (10.1016/j.nut.2013.09.008) Copyright © 2014 Elsevier Inc. Terms and Conditions

Fig. 6 Changes in the percentage of (A) CD8+ and (C) CD8+ TCRγδ+ IELs among the pups of the VAN, VAD, and VAD-N groups in the presence or absence of LPS challenge (n = 5 to 7). The VA significant main effect, independent of LPS treatment, on the percentage of (B) CD8+ and (D) CD8+ TCRγδ+ IELs among the three combined VAN, VAD and VAD-N groups. “Interaction” indicates an effect of LPS treatment versus null treatment. *P < 0.05; †P < 0.01; ‡P < 0.001; n.s.= not significant in post hoc tests. IELs, intestinal intraepithelial lymphocytes; LPS, lipopolysaccharide; TCR, T-cell antigen receptor; VAD, vitamin A deficiency; VAD-N, VAD during gestation and fed the milk of VAN rats after birth; VAN, vitamin A normal. Nutrition 2014 30, 350-357DOI: (10.1016/j.nut.2013.09.008) Copyright © 2014 Elsevier Inc. Terms and Conditions