Decrypting drug actions and protein modifications by dose- and time-resolved proteomics.

Although most cancer drugs modulate the activities of cellular pathways by changing posttranslational modifications (PTMs), little is known regarding the extent and the time- and dose-response characteristics of drug-regulated PTMs. In this work, we introduce a proteomic assay called decryptM that quantifies drug-PTM modulation for thousands of PTMs in cells to shed light on target engagement and drug mechanism of action. Examples range from detecting DNA damage by chemotherapeutics, to identifying drug-specific PTM signatures of kinase inhibitors, to demonstrating that rituximab kills CD20-positive B cells by overactivating B cell receptor signaling. DecryptM profiling of 31 cancer drugs in 13 cell lines demonstrates the broad applicability of the approach. The resulting 1.8 million dose-response curves are provided as an interactive molecular resource in ProteomicsDB.

Modulation of the Systemic Immune Response in Suckling Rats by Breast Milk TGF-ß2, EGF and FGF21 Supplementation.

Breast milk is a rich fluid containing bioactive compounds such as specific growth factors (GF) that contribute to maturation of the immune system in early life. The aim of this study was to determine whether transforming growth factor-ß2 (TGF-ß2), epidermal growth factor (EGF) and fibroblast growth factor 21 (FGF21), compounds present in breast milk, could promote systemic immune maturation. For this purpose, newborn Wistar rats were daily supplemented with these GF by oral gavage during the suckling period (21 days of life). At day 14 and 21 of life, plasma for immunoglobulin (Ig) quantification was obtained and spleen lymphocytes were isolated, immunophenotyped and cultured to evaluate their ability to proliferate and release cytokines. The main result was obtained at day 14, when supplementation with EGF increased B cell proportion to reach levels observed at day 21. At the end of the suckling period, all GF increased the plasma levels of IgG1 and IgG2a isotypes, FGF21 balanced the Th1/Th2 cytokine response and both EGF and FGF21 modified splenic lymphocyte composition. These results suggested that the studied milk bioactive factors, mainly EGF and FGF21, may have modulatory roles in the systemic immune responses in early life, although their physiological roles remain to be established.

TGF-ß2, EGF, and FGF21 Growth Factors Present in Breast Milk Promote Mesenteric Lymph Node Lymphocytes Maturation in Suckling Rats.

Breast milk, due to its large number of nutrients and bioactive factors, contributes to optimal development and immune maturation in early life. In this study, we aimed to assess the influence of some growth factors present in breast milk, such as transforming growth factor-ß2 (TGF-ß2), epidermal growth factor (EGF), and fibroblast growth factor 21 (FGF21), on the immune response development. Newborn Wistar rats were supplemented daily with TGF-ß2, EGF, or FGF21, throughout the suckling period. At day 14 and 21 of life, lymphocytes from mesenteric lymph nodes (MLNs) were isolated, immunophenotyped, and cultured to evaluate their ability to proliferate and release cytokines. The main results demonstrated that supplementation with TGF-ß2, EGF, or FGF21 modified the lymphocyte composition in MLNs. At day 14, all supplementations were able to induce a lower percentage of natural killer (NK) cells with the immature phenotype (CD8⁺), and they reduced the CD8αα/CD8αß ratio at day 21. Moreover, the cytokine pattern was modified by the three treatments, with a down regulation of interleukin (IL)-13 secretion. These results showed the contribution of these growth factors in the lymphocytes MLNs immune maturation during the neonatal period.

Leptin and adiponectin supplementation modifies mesenteric lymph node lymphocyte composition and functionality in suckling rats.

At birth, when immune responses are insufficient, there begins the development of the defence capability against pathogens. Leptin and adiponectin, adipokines that are present in breast milk, have been shown to play a role in the regulation of immune responses. We report here, for the first time, the influence of in vivo adipokine supplementation on the intestinal immune system in early life. Suckling Wistar rats were daily supplemented with leptin (0·7 µg/kg per d, n 36) or adiponectin (35 µg/kg per d, n 36) during the suckling period. The lymphocyte composition, proliferation and cytokine secretion from mesenteric lymph node lymphocytes (on days 14 and 21), as well as intestinal IgA and IgM concentration (day 21), were evaluated. At day 14, leptin supplementation significantly increased the TCRαß + cell proportion in mesenteric lymph nodes, in particular owing to an increase in the TCRαß + CD8+ cell population. Moreover, the leptin or adiponectin supplementation promoted the early development CD8+ cells, with adiponectin being the only adipokine capable of enhancing the lymphoproliferative ability at the end of the suckling period. Although leptin decreased intestinal IgA concentration, it had a trophic effect on the intestine in early life. Supplementation of both adipokines modulated the cytokine profile during (day 14) and at the end (day 21) of the suckling period. These results suggest that leptin and adiponectin during suckling play a role in the development of mucosal immunity in early life.

Cocoa Diet Prevents Antibody Synthesis and Modifies Lymph Node Composition and Functionality in a Rat Oral Sensitization Model.

Cocoa powder, a rich source of polyphenols, has shown immunomodulatory properties in both the intestinal and systemic immune compartments of rats. The aim of the current study was to establish the effect of a cocoa diet in a rat oral sensitization model and also to gain insight into the mesenteric lymph nodes (MLN) activities induced by this diet. To achieve this, three-week-old Lewis rats were fed either a standard diet or a diet with 10% cocoa and were orally sensitized with ovalbumin (OVA) and with cholera toxin as a mucosal adjuvant. Specific antibodies were quantified, and lymphocyte composition, gene expression, and cytokine release were established in MLN. The development of anti-OVA antibodies was almost totally prevented in cocoa-fed rats. In addition, this diet increased the proportion of TCRγδ+ and CD103+CD8+ cells and decreased the proportion of CD62L+CD4+ and CD62L+CD8+ cells in MLN, whereas it upregulated the gene expression of OX40L, CD11c, and IL-1ß and downregulated the gene expression of IL-17α. In conclusion, the cocoa diet induced tolerance in an oral sensitization model accompanied by changes in MLN that could contribute to this effect, suggesting its potential implication in the prevention of food allergies.

Impact of cocoa polyphenol extracts on the immune system and microbiota in two strains of young rats.

A diet containing 10% cocoa, a rich source of polyphenols and fibre, is able to modify intestinal immune status as well as microbiota composition. The present study was aimed at investigating whether cocoa flavonoid content is uniquely responsible for these modulatory effects of cocoa, and to establish whether these effects depend on the rat strain. To this end, 3-week-old Wistar and Brown Norway rats were fed, for 4 weeks, either a standard diet or the following three isoenergetic diets containing increasing proportions of cocoa flavonoids from different sources: one with 0.2% polyphenols (from conventional defatted cocoa), and two others with 0.4 and 0.8% polyphenols (from non-fermented cocoa, very rich in polyphenols). Serum Ig concentrations, faecal IgA levels, microbiota composition and IgA-coating bacterial proportion were evaluated at the beginning and at the end of the study. After the nutritional intervention, the composition of lymphocytes in Peyer's patches and mesenteric lymph nodes was evaluated. In some respects, the Wistar strain was more sensitive to the impact of the cocoa diets than the Brown Norway strain. After 4 weeks of dietary intervention, similar modulatory effects of the diets containing 0.2 and 0.8% polyphenols on mucosal IgA levels and microbiota composition were found, although the 0.2% diet, with a higher proportion of theobromine and fibre, had more impact, suggesting that polyphenols are not the only components involved in such effects.

A diet enriched with cocoa prevents IgE synthesis in a rat allergy model.

Previous studies in young rats reported the impact of cocoa intake on healthy immune status and allow suggesting it may have a role in the prevention of some immune-mediated diseases. The aim of this study was to ascertain the effect of a cocoa diet in a model of allergy in young rats. Three-week-old Brown Norway rats were immunized by i.p. injection of ovalbumin (OVA) with alum as adjuvant and Bordetella pertussis toxin. During the next 4 weeks rats received either a cocoa diet (containing 0.2% polyphenols, w/w) or a standard diet. Animals fed a standard diet showed high concentrations of anti-OVA IgG1, IgG2a, IgG2b and high anti-OVA IgE titres, which is the antibody involved in allergic response. In contrast, animals fed a cocoa diet showed significantly lower concentrations of anti-OVA IgG1 and IgG2a antibodies. Interestingly, the cocoa diet prevented anti-OVA IgE synthesis and decreased total serum IgE concentration. Analysis of cytokine production in lymph node cells at the end of the study revealed that, in this compartment, the cocoa diet decreased the tumor necrosis factor (TNF)-α and the interleukin (IL)-10 secretion but not IL-4 production. In conclusion, a cocoa-enriched diet in young rats produces an immunomodulatory effect that prevents anti-allergen IgE synthesis, suggesting a potential role for cocoa flavonoids in the prevention or treatment of allergic diseases.