QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: 139/1/69    most recent jn.108.092528v1 Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ozawa, T. Articles by Nakao, A. Search for Related Content PubMed PubMed Citation Articles by Ozawa, T. Articles by Nakao, A.

---

Nutritional Immunology

Transforming Growth Factor-β Activity in Commercially Available Pasteurized Cow Milk Provides Protection against Inflammation in Mice^1,2

³ Department of Immunology and ⁴ Department of Human Pathology, Faculty of Medicine, University of Yamanashi, Yamanashi 409-3898, Japan; and ⁵ Atopy Research Center, Juntendo University School of Medicine, Tokyo 113-8421, Japan

^* To whom correspondence should be addressed. E-mail: anakao{at}yamanashi.ac.jp.

	ABSTRACT

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
Cow milk contains a large amount of an immunoregulatory cytokine, transforming growth factor-β (TGFβ). The present study investigated whether commercially available pasteurized cow milk retains TGFβ activity both in vitro and in vivo. Some commercial cow milk increased TGFβ/Smad-responsive reporter activity and induced Smad2 phosphorylation and the transcription of the TGFβ/Smad target genes TGFβ itself and Smad7 in vitro. Mice treated orally with 500 µL of cow milk containing TGFβ (3 µg/L) daily for 2 wk had increased phosphorylation of Smad2 and TGFβ and Smad7 mRNA expression in the intestine. These mice also had significantly greater serum TGFβ concentrations than the mice treated orally with PBS. Furthermore, oral administration of 500 µL of cow milk containing TGFβ (3 µg/L) daily for 2 wk before the induction of dextran sodium sulfate colitis and lipopolysaccharide-induced endotoxemia ameliorated tissue damage and mortality, respectively, in mice. These in vivo effects of cow milk were abrogated by the simultaneous administration of TGFβ type I receptor kinase inhibitor with the cow milk, and they were not observed after the oral administration of cow's milk containing little TGFβ. In humans, 1 oral challenge of 10 mL/kg cow milk containing TGFβ (3 µg/L) increased the plasma TGFβ concentrations at 4 h after the challenge. Thus, some commercially available pasteurized cow milk retains TGFβ activity, which may be able to provide protection against experimental colitis and endotoxemia associated with increased intestinal and circulating TGFβ levels.

	Introduction

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

TGFβ is secreted as inactive (latent) precursors into the extracellular space and it requires an activation process involving extreme pH to mediate its biological activity (4). TGFβ principally signals through 2 serine/threonine kinase receptors (termed type I and type II) and the Smad family of proteins (5). TGFβ1 binds directly to the type II receptor, whereas binding of TGFβ2 to the type II receptor requires coexpression of the type I receptor or the TGFβ type III receptor (β-glycan), which is a membrane-bound proteoglycan with a short cytoplasmic tail that has no apparent signaling motif (6). The type I receptor is activated by the type II receptor upon ligand binding and induces the phosphorylation of Smad2 and Smad3, which in turn forms complexes with Smad4. The Smad complexes then move into the nucleus and regulate the transcriptional responses of the TGFβ target genes such as Smad7.

Cow milk contains not only essential nutrients but also a number of natural bioactive substances that have many beneficial effects on human health (7–10). Raw cow milk contains a large amount of TGFβ, with a predominance of TGFβ2 over TGFβ1 (10,11); some of the beneficial effects of cow milk or cow milk-derived whey proteins on human health, particularly on inflammatory activity, have been suggested to be associated with this immunoregulatory molecule (10). However, it remains largely unclear whether commercially available cow milk still retains TGFβ activity following pasteurization and manufacturing processes and whether the commercial cow milk-borne TGFβ activity, if any, has inhibitory effects on inflammatory diseases.

The aim of this study was therefore to clarify TGFβ activity in commercially available cow milk and its efficacy in reducing inflammation. For this purpose, we first examined the TGFβ activity in commercially available cow milk in vitro and in vivo and then investigated whether this activity provided protection against experimental colitis and endotoxemia in mice.

	Materials and Methods

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
    Mice. Female 4- to 6-wk-old BALB/c mice were purchased from SLC and were bred under specific pathogen-free conditions. All animal experiments were approved by the Institutional Review Board of the University of Yamanashi.

    Commercial cow milk samples. Seven cow milk samples that were commercially available in Japan were purchased from the following companies: 1, Meiji Nyugyo; 2, Yatsugatake Nyugyo; 3, Morinaga Nyugyo; 4, Shizuoka Gyunyu Coop.; 5, Glico Nyugyo (no. 1); 6, Japan Milk Committee; 7, Glico Nyugyo Inc. (no. 2). Samples were immediately used for later analysis. For some experiments, TGF-β in milk was activated at room temperature by dropwise addition of HCL (5 mol/L) to pH 2.0, neutralized with NaOH (5 mol/L), and diluted with PBS.

    Cell culture. The human alveolar carcinoma cell line A549 and colon epithelial cell line Caco2 (RIKEN Cell Bank, Ibaragi, Japan) were maintained in DMEM medium (Invitrogen/Gibco) containing 10% fetal calf serum and antibiotics.

    Transcriptional reporter assay. A549 cells were seeded at 3 x 10⁴ per well in 24-well plates. The cells were then transfected with 200 ng of (CAGA)₁₂-luciferase reporter plasmid (CAGA₁₂-luc), which is exclusively activated by the TGFβ-induced complex between Smad3 and Smad4 (12), and 5 ng of pRL-TK Renilla luciferase vector (Promega), an internal control for transfection efficiency, using FuGENE 6 transfection reagent (Roche Diagnostics). After 12 h, the cells were stimulated with 500 µL of milk sample 2 or 7 (the estimated concentration of total TGFβ2 in cow milk sample 2 was 3 µg/L based on ELISA results) (Fig. 1A) or 10 µg/L TGFβ1 (R & D Inc.) with or without 10 µmol/L HTS466284 (HTS), a selective small molecule inhibitor of TGFβ type I receptor kinase (13) (Calbiochem) or 10 mg/L anti-TGFβ1/2/3 neutralizing antibody (1D11) or anti-tumor necrosis factor- (TNF) neutralizing antibody (R & D Inc.) Twelve hours after the stimulation, the firefly and Renilla luciferase activities were measured as previously described (12).

View larger version (29K): [in this window] [in a new window]   FIGURE 1  Commercial cow milk retains TGFβ activity in vitro. (A) The concentrations of TGFβ2 in 7 commercial cow milk samples with or without acid pretreatment. (B,C) (CAGA)12 luc-reporter activity induced by nontreated or acid-treated cow milk sample 2 in A549 cells with or without HTS (B) or with or without anti-TGFβ (TGFβ) or anti-TNF antibody (TNF) (C). (D) Expression of phosphorylated Smad2 (pSmad2), Smad2/3 (Smad2/3), or β-actin (actin) in A549 cells stimulated with the acid-treated cow milk sample 2 or TGFβ for the indicated times with or without HTS. (E) Expression of Smad7 mRNA in Caco2 cells stimulated with nontreated cow milk sample 2 or TGFβ for the indicated times with or without HTS. Values are means ± SD, n = 3. *Means differ, P < 0.05.

    Oral administration of commercial cow milk. To determine the in vivo activity of TGFβ in commercial cow milk, mice were administered 500 µL (250 µL x 2 times per day) of cow milk sample 2 (1.5 ng total TGFβ2 per mouse), sample 7, or control PBS by gastric intubation daily for 14 d. Six hours after the final challenge, the mice were anesthetized with an overdose of Halothane and killed by cervical dislocation for the subsequent analysis (histology, real-time PCR, and ELISA). For some experiments, HTS (10 mg/kg body weight) or a control vehicle dimethylsulfoxide (DMSO) were i.v. administered simultaneously with cow milk sample 2 every other day for a total of 7 times. The dosages of HTS (10 mg/kg body weight) were chosen based on the findings in our previous experiment (15).

    Histology. Mouse jejunum and colon were removed, fixed with 4% paraformaldehyde in PBS, and embedded in paraffin. The tissue sections were stained with hematoxylin and eosin. For immunohistochemistry, the sections were deparaffinized, stained with anti-phosphorylated Smad2 antibody (Santa Cruz Biotechnology), and visualized by the use of peroxidase-based VECTASTAIN ABC kits with DAB substrate (Vector Laboratories) as previously described (16).

    Assessment of histological examination. The number of phosphorylated Smad2-positive cells in the intestinal sections was counted microscopically without knowledge of the treatments. Briefly, a minimum of 100 cells in the intestinal epithelium was counted in a high power field (x400) for each sample. The percentage of phosphorylated Smad2-positive cells in the total intestinal epithelial cells was expressed (percent). Two or 4 specimens of the phosphorylated Smad2-stained intestinal sections from 1 mouse were selected. The mean score was calculated and then the mean scores were calculated in 6 mice.

    Quantitative real-time PCR. Quantitative PCR analysis using cDNA from the mouse tissue specimens was performed using the AB7500 real-time PCR system (Applied Biosystems) according to the manufacturer's instructions using primers and probes for human TGFβ2, Smad7, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Applied Biosystems) as previously described (16). The ratio of each gene (TGFβ2 and Smad7) to that of GAPDH was calculated and the relative expression levels are shown.

    ELISA. The amounts of TGFβ1, β2, and TNF in the mouse serum or human plasma were determined using the mouse or human TGFβ1, TGFβ2, or mouse TNF ELISA kits (R&D Inc.) according to the manufacturer's instructions. To determine the total TGFβ1 or TGFβ2 concentrations in the mouse serum and human plasma, the samples were activated by an acidification procedure prior to the ELISA assay.

    Induction of colitis. Mice were pretreated orally with either PBS or cow milk sample 2 or 7 every day for 14 d with or without i.v. administration of HTS as described above and then colitis was induced by feeding 3% dextran sodium sulfate (DSS) dissolved in drinking distilled water (molecular weight, 5000; Wako Pure Chemical) as described previously (17). The degree of inflammation was histologically scored as previously described (17). Briefly, scores of inflammation were as follows: 0, no increased inflammatory infiltrates; 1, focal mild inflammation; 2, diffuse mild inflammation; 3, cyptic abscess formation; and 4, diffuse dense inflammation.

    Induction of endotoxemia. Mice were pretreated orally with PBS or cow milk sample 2 or 7 everyday for 14 d with or without i.v. administration of HTS as described above and then the mice were injected intraperitoneally with 5–10 mg/kg lipopolysaccharide (LPS) (Sigma Aldrich). We observed survival of mice to 7–10 d as previously described (18). For analysis of cytokine production, blood from live mice was collected at 48 h after the LPS injection and then the serum specimens were collected.

    Human subjects. Five healthy adult volunteers (3 women and 2 men, aged 24–45 y) drank 10 mL/kg cow milk sample 2. Before and 4 h after drinking the milk, plasma samples were obtained from each person. Participants were allowed to drink only water during the 12-h period before the collection of the plasma samples. The protocol was approved by the Ethical Committee of University of Yamanashi. The volunteers gave written informed consent.

    Statistical analysis. The data are summarized as the means ± SD. Differences between 2 groups for in vitro assays and for TGFβ concentrations in human plasma were determined using the paired Student's t-test. Differences among multiple groups for mouse experiments were determined by the Kruskal-Wallis test. The statistical analysis of mouse survival data was performed using the Logrank test. P < 0.05 was considered significant. The statistical analysis was performed using Prism (GraphPad Software).

	Results

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
    Commercial cow milk containing TGFβ retains its activity in vitro. Six of the 7 commercially available cow milk samples contained a large amount of the active form of TGFβ2 (Fig. 1A). The variability in the concentrations may have been due to differences in the pasteurization methods and manufacturing processes. Transient acidification increased the TGFβ2 concentrations in the milk samples (Fig. 1A). Because milk sample 2 had the highest concentration of TGFβ2 (1.5 µg/L, active form), whereas sample 7 contained little TGFβ2, these samples were used for the subsequent experiments. Without acidification, milk sample 2 significantly increased the luciferase activity of the TGFβ/Smad reporter construct CAGA₁₂-luc, which was enhanced by acidification (Fig. 1B). The increases in the luciferase activity by the nontreated or acid-treated milk sample 2 were comparable to those achieved by theoretically equivalent concentrations of recombinant TGFβ based on the ELISA data (data not shown). Importantly, the addition of HTS or anti-TGFβ neutralizing antibody, but not anti-TNF neutralizing antibody, completely suppressed the milk sample 2-induced increase in luciferase activity (Fig. 1B,C), thus suggesting the increased luciferase activity depended on the TGFβ activity in cow milk. In addition, milk sample 2 induced the phosphorylation of Smad2, which was also blocked by HTS (Fig. 1D). In the human colon epithelial cell line Caco2, milk sample 2 induced mRNA expression of TGFβ2 (data not shown) and Smad7 (Fig. 1E). The milk sample 2-induced Smad7 mRNA was inhibited by HTS (Fig. 1E). Collectively, some of the commercially available cow milk, even after pasteurization and manufacturing processes, retained a substantial degree of TGFβ activity in vitro.

    Commercial cow milk containing TGFβ retains its activity in vivo. To determine whether commercial cow milk containing TGFβ retains its activity in vivo, the induction of Smad2 phosphorylation and the transcriptional response of TGFβ target genes TGFb itself (19) and Smad7 (20) in mouse intestinal tissue were next assessed.

The immunoreactivity of phosphorylated Smad2 in the small and large intestinal tissue was upregulated after the oral administration of the cow milk sample 2, but not sample 7, containing TGFβ (Fig. 2A,B; Table 1). The phosphorylated Smad2-positive cells were predominately intestinal epithelial cells with nuclear staining. Similarly, the expression of TGFβ2 and Smad7 mRNA was significantly induced following oral administration of cow milk sample 2, but not 7, in the small and large intestine (Fig. 3A–H). Treatment of mice with HTS during the milk feeding inhibited the milk-induced phosphorylation of Smad2 and TGFβ2 and Smad7 mRNA expression in the mouse intestine (Figs. 2A,B and 3A,B,E,F; Table 1). Interestingly, the concentrations of serum TGFβ2 significantly increased after oral administration of cow milk sample 2, but not 7, which was again blocked by treatment with HTS during the milk feeding (Fig. 3I,J). These results suggest that the oral administration of commercial cow milk containing TGFβ can activate TGFβ/Smad signaling in the mouse intestine and upregulate intestinal and circulating TGFβ2 concentrations, depending on TGFβ activity in cow milk.

View larger version (86K): [in this window] [in a new window]   FIGURE 2  Commercial cow milk containing TGFβ induces phosphorylation of Smad2 in the mouse intestine. Expression of phosphorylated Smad2 (pSmad2 immuno-stain) of the mouse jejunum (A) or colon (B) tissue sections obtained from the mice treated orally with PBS or cow milk sample 2 or 7 with or without HTS or control DMSO. Positive staining is indicated as a brown color. HE stain: staining with hematoxylin and eosin solution.

View larger version (14K): [in this window] [in a new window]   FIGURE 3  Commercial cow milk containing TGFβ induces expression of TGFβ target genes in the mouse intestine. (A–H) Expression of TGFβ2, Smad7, and GAPDH mRNA in the jejunum (A–D) or the colon (E–H) tissue samples obtained from the mice treated orally with PBS or cow milk sample 2 or 7 with or without HTS or control DMSO. (I,J) TGFβ2 concentrations in the mouse serum obtained from the mice treated above. Values are means ± SD, n = 6. Labeled means without a common letter differ, P < 0.05.

Mice pretreated with control PBS or cow milk sample 7 had weight loss, bloody stool, and colonic inflammation after the administration of DSS (Fig. 4A–C; data not shown). In contrast, these changes did not occur in mice pretreated with cow milk sample 2 containing TGFβ. Importantly, the simultaneous administration of HTS with cow milk sample 2 abrogated the protective effects against DSS-induced colitis (Fig. 4A–C). The serum TGFβ2 concentrations significantly increased after oral administration of cow milk at the point of evaluation for colitis, which was blocked by treatment with HTS during the milk feeding (Fig. 4D,E).

View larger version (27K): [in this window] [in a new window]   FIGURE 4  Commercial cow milk containing TGFβ provides protection against DSS-induced colitis in mice. (A,B) The weight changes from the baseline during the DSS treatment. *P < 0.05. (C) The scoring of inflammation in the colons of the mice at d 8 postadministration of 3% DSS. (D,E) The serum TGFβ2 concentrations at d 8 postadministration of 3% DSS. Values are means ± SD, n = 6. Labeled means without a common letter differ, P < 0.05.

View larger version (13K): [in this window] [in a new window]   FIGURE 5  Commercial cow milk containing TGFβ provides protection against LPS-induced lethality in mice. (A,B) Survival rates in the mice pretreated with PBS or cow milk sample 2 or 7 before injection with a lethal dose of LPS, n = 10. *P < 0.05.

View this table: [in this window] [in a new window]   TABLE 2 TNF concentrations in the mouse serum1

View larger version (10K): [in this window] [in a new window]   FIGURE 6  Oral administration of cow milk increases TGFβ2 concentrations in human plasma. The concentrations of total TGFβ2 (A) and TGFβ1 (B) in plasma samples obtained before (0 h) and 4 h after the intake of 10 mL/kg of cow milk sample 2 containing TGFβ. n = 5; *P < 0.05.

	Discussion

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

Raw cow milk or human milk from lactating mothers contains a large amount of TGFβ (10,11). However, it remains largely unclear whether commercially available cow milk still retains TGFβ activity following pasteurization and manufacturing processes. We found that some of the commercially available cow milk still contained TGFβ2 (0.3–3 µg/L of total TGFβ2; Fig. 1) to a similar extent to that found in raw cow milk (4.3 µg/L of total TGFβ) (11) and in human milk (3.1 µg/L of total TGFβ2 at 2 wk of lactation) (25). McPherson et al. (26) previously reported that the concentrations of TGFβ2 in whole milk from lactating mothers were well preserved after holder pasteurization at 56.5°C. Our results showing preserved TGFβ activity as well as expression in commercially available cow milk are consistent with their findings.

Approximately one-half of the total TGFβ was already activated in commercially available cow milk (Fig. 1A). Because heat can activate the latent form of TGFβ (4), the presence of the active form of TGFβ in commercially available cow milk may be due to pasteurization. It remains to be determined what factors in the manufacturing processes influence the activation and retention of TGFβ in commercial cow milk.

Because cow milk contains many bioactive molecules other than TGFβ (27), the in vivo effects of cow milk observed in this study might not be mediated by TGFβ alone present in cow milk. However, the in vitro and in vivo intervention studies with anti-TGFβ neutralizing antibody, TGFβ receptor kinase inhibitor, and the comparison with cow milk containing little TGFβ (Figs. 1–5) strongly suggest that the abilities of cow milk to affect TGFβ signaling/expression and provide protection against experimental colitis and endotoxemia are primarily mediated by TGFβ, which is already present in cow milk.

Because endogenous TGFβ activity was shown to protect against DSS-induced colitis and LPS-induced endotoxemia/shock in rodents (17,21,23), it is likely that the increased circulating TGFβ concentrations by cow milk is directly responsible for the protection. However, because TGFβ has multiple roles in immunoregulation, including the induction of regulatory T cells (8,28), other immunological mechanisms may be involved in such protection.

We and others have recently shown that the oral administration of TGFβ induces the activation of intestinal TGFβ/Smad signaling, enhances oral tolerance, and inhibits allergic reaction to an oral antigen in rodents (16,29,30). In addition, Verhasselt et al. (31) reported that breast milk-mediated transfer of an antigen induces tolerance and protection from allergic asthma in neonates via TGFβ present in breast milk. These and the current findings support the notion that orally administered TGFβ, such as TGFβ in cow milk, can still retain TGFβ activity in the hostile gastrointestinal environment where degradation of this protein by gastric acid or proteases in the intestine can occur and has the ability to modulate mucosal and systemic immune responses. It would be, therefore, attractive to speculate that a daily food product containing TGFβ, such as cow milk, may insidiously modulate mucosal and systemic immune responses, thereby providing some beneficial effects on human health in the long term.

In conclusion, we suggest that TGFβ activity found in commercially available cow milk can provide protection against inflammation. It remains to be determined whether the current findings using 2 commercial cow milk samples can be generalized to include all brands of commercial cow milk. In Western societies, the consumption of milk has decreased partly due to some reported negative health effects, such as allergic reaction to the contents of milk. However, the current results may provide a new rationale to support the benefits of cow milk in human health, addressing a decisive regulatory factor required for the positive health effects of cow milk.

	FOOTNOTES

 
¹ Supported in part by grants from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.

² Author disclosures: T. Ozawa, M. Miyata, M. Nishimura, T. Ando, Y. Ouyang, T. Ohba, N. Shimokawa, Y. Ohnuma, R. Katoh, H. Ogawa, and A. Nakao, no conflicts of interest.

⁶ Abbreviations used: DMSO, dimethylsulfoxide; DSS, dextran sodium sulfate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HTS, HTS466284; LPS, lipopolysaccharide; TGFβ, transforming growth factor-β; TNF, tumor necrosis factor-.

Manuscript received 7 May 2008. Initial review completed 5 June 2008. Revision accepted 16 October 2008.

	LITERATURE CITED

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 

1. Li MO, Wan YY, Sanjabi S, Robertson AK, Flavell RA. Transforming growth factor-β regulation of immune responses. Annu Rev Immunol. 2006;24:99–146.[CrossRef][Medline]

2. Massagué J. The transforming growth factor-β family. Annu Rev Cell Biol. 1990;6:597–641.[CrossRef][Medline]

3. Cheifetz S, Hernandez H, Laiho M, ten Dijke P, Iwata KK, Massagué J. Distinct transforming growth factor-β (TGFβ) receptor subsets as determinants of cellular responsiveness to three TGFβ isoforms. J Biol Chem. 1990;265:20533–8.[Abstract/Free Full Text]

4. Annes JP, Munger JS, Rifkin DB. Making sense of latent TGFβ activation. J Cell Sci. 2003;116:217–24.[Abstract/Free Full Text]

5. Schmierer B, Hill CS. TGFβ-SMAD signal transduction: molecular specificity and functional flexibility. Nat Rev Mol Cell Biol. 2007;8:970–82.[CrossRef][Medline]

6. Rodriguez C, Chen F, Weinberg RA, Lodish HF. Cooperative binding of transforming growth factor (TGF)-β2 to the types I and II TGFβ receptors. J Biol Chem. 1995;270:15919–22.[Abstract/Free Full Text]

7. Murphy MS. Growth factors and the gastrointestinal tract. Nutrition. 1998;14:771–4.[CrossRef][Medline]

8. Meisel H. Biochemical properties of peptides encrypted in bovine milk proteins. Curr Med Chem. 2005;12:1905–19.[CrossRef][Medline]

9. Gill HS, Doull F, Rutherfurd KJ, Cross ML. Immunoregulatory peptides in bovine milk. Br J Nutr. 2000;84:S111–7.[Medline]

10. Donnet-Hughes A, Duc N, Serrant P, Vidal K, Schiffrin EJ. Bioactive molecules in milk and their role in health and disease: the role of transforming growth factor-β. Immunol Cell Biol. 2000;78:74–9.[CrossRef][Medline]

11. Rogers ML, Goddard C, Regester GO, Ballard FJ, Belford DA. Transforming growth factor-β in bovine milk: concentration, stability and molecular mass forms. J Endocrinol. 1996;151:77–86.[Abstract/Free Full Text]

12. Dennler S, Itoh S, Vivien D, ten Dijke P, Huet S, Gauthier JM. Direct binding of Smad3 and Smad4 to critical TGFβ-inducible elements in the promoter of human plasminogen activator inhibitor-type 1 gene. EMBO J. 1998;17:3091–100.[CrossRef][Medline]

13. Sawyer JS, Anderson BD, Beight DW, Campbell RM, Jones ML, Herron DK, Lampe JW, McCowan JR, McMillen WT, et al. Synthesis and activity of new aryl- and heteroaryl-substituted pyrazole inhibitors of the transforming growth factor-β type I receptor kinase domain. J Med Chem. 2003;46:3953–6.[CrossRef][Medline]

14. Kanamaru Y, Sumiyoshi K, Ushio H, Ogawa H, Okumura K, Nakao A. Smad3 deficiency in mast cells provides efficient host protection against acute septic peritonitis. J Immunol. 2005;174:4193–7.[Abstract/Free Full Text]

15. Sakuma M, Hatsushika K, Koyama K, Katoh R, Ando T, Watanabe Y, Wako M, Kanzaki M, Takano S, et al. TGFβ type I receptor kinase inhibitor down-regulates rheumatoid synoviocytes and prevents the arthritis induced by type II collagen antibody. Int Immunol. 2007;19:117–26.[Abstract/Free Full Text]

16. Ando T, Hatsushika K, Wako M, Ohba T, Koyama K, Ohnuma Y, Katoh R, Ogawa H, Okumura K, et al. Orally administered TGFβ is biologically active in the intestinal mucosa and enhances oral tolerance. J Allergy Clin Immunol. 2007;120:916–23.[Medline]

17. Hahm KB, Im YH, Parks TW, Park SH, Markowitz S, Jung HY, Green J, Kim SJ. Loss of transforming growth factor-β signalling in the intestine contributes to tissue injury in inflammatory bowel disease. Gut. 2001;49:190–8.[Abstract/Free Full Text]

18. Sha T, Sunamoto M, Kitazaki T, Sato J, Ii M, Iizawa Y. Therapeutic effects of TAK-242, a novel selective Toll-like receptor 4 signal transduction inhibitor, in mouse endotoxin shock model. Eur J Pharmacol. 2007;571:231–9.[CrossRef][Medline]

19. Kim SJ, Jeang KT, Glick AB, Sporn MB, Roberts AB. Promoter sequences of the human transforming growth factor-β1 gene responsive to transforming growth factor-β1 autoinduction. J Biol Chem. 1989;264:7041–5.[Abstract/Free Full Text]

20. Nakao A, Afrakhte M, Morén A, Nakayama T, Christian JL, Heuchel R, Itoh S, Kawabata M, Heldin NE, et al. Identification of Smad7, a TGFβ-inducible antagonist of TGFβ signalling. Nature. 1997;389:631–5.[CrossRef][Medline]

21. Sakuraba H, Ishiguro Y, Yamagata K, Munakata A, Nakane A. Blockade of TGFβ accelerates mucosal destruction through epithelial cell apoptosis. Biochem Biophys Res Commun. 2007;359:406–12.[Medline]

22. Perrella MA, Hsieh CM, Lee WS, Shieh S, Tsai JC, Patterson C, Lowenstein CJ, Long NC, Haber E, et al. Arrest of endotoxin-induced hypotension by transforming growth factor-β1. Proc Natl Acad Sci USA. 1996;93:2054–9.[Abstract/Free Full Text]

23. McCartney-Francis N, Jin W, Wahl SM. Aberrant Toll receptor expression and endotoxin hypersensitivity in mice lacking a functional TGFβ1 signaling pathway. J Immunol. 2004;172:3814–21.[Abstract/Free Full Text]

24. Van Amersfoort ES, Van Berkel TJ, Kuiper J. Receptors, mediators, and mechanisms involved in bacterial sepsis and septic shock. Clin Microbiol Rev. 2003;16:379–414.[Abstract/Free Full Text]

25. Hawkes JS, Bryan DL, James MJ, Gibson RA. Cytokines (IL-1β, IL-6, TNF, TGFβ1, and TGFβ2) and prostaglandin E2 in human milk during the first three months postpartum. Pediatr Res. 1999;46:194–9.[Medline]

26. McPherson RJ, Wagner CL. The effect of pasteurization on transforming growth factor- and transforming growth factor-β2 concentrations in human milk. Adv Exp Med Biol. 2001;501:559–63.[Medline]

27. Goldman AS, Chheda S, Garofalo R. Evolution of immunologic functions of the mammary gland and the postnatal development of immunity. Pediatr Res. 1998;43:155–62.[Medline]

28. Wahl SM, Wen J, Moutsopoulos N. TGFβ: a mobile purveyor of immune privilege. Immunol Rev. 2006;213:213–27.[CrossRef][Medline]

29. Okamoto A, Kawamura T, Kanbe K, Kanamaru Y, Ogawa H, Okumura K, Nakao A. Suppression of serum IgE response and systemic anaphylaxis in a food allergy model by orally administered high-dose TGFβ. Int Immunol. 2005;17:705–12.[Abstract/Free Full Text]

30. Penttila I. Effects of transforming growth factor-β and formula feeding on systemic immune responses to dietary beta-lactoglobulin in allergy-prone rats. Pediatr Res. 2006;59:650–5.[CrossRef][Medline]

31. Verhasselt V, Milcent V, Cazareth J, Kanda A, Fleury S, Dombrowicz D, Glaichenhaus N, Julia V. Breast milk-mediated transfer of an antigen induces tolerance and protection from allergic asthma. Nat Med. 2008;14:170–80.[Medline]

This article has been cited by other articles:

				Y. Nakamura, M. Miyata, T. Ando, N. Shimokawa, Y. Ohnuma, R. Katoh, H. Ogawa, K. Okumura, and A. Nakao The Latent Form of Transforming Growth Factor-{beta} Administered Orally Is Activated by Gastric Acid in Mice J. Nutr., August 1, 2009; 139(8): 1463 - 1468. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 139/1/69    most recent jn.108.092528v1 Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ozawa, T. Articles by Nakao, A. Search for Related Content PubMed PubMed Citation Articles by Ozawa, T. Articles by Nakao, A.