Probiotic Bifidobacterium lactis, anti-oxidant vitamin E/C and anti-inflammatory dha attenuate lung inflammation due to pm2.5 exposure in mice.

The incidence of asthma and allergic diseases of the airways is constantly increasing, both in the industrialised and developing countries, due to harmful and excessive quantities of air pollution. Although some studies have shown an effect of dietary supplementation of specific nutrients (especially with anti-oxidant and anti-inflammatory properties) in reducing airways inflammatory response, the results are not yet conclusive and the science is still at its infancy. Our hypothesis is that combining such nutrients could provide more benefits than using them alone. The aim of the research project proposed here is to investigate whether specific combinations of nutrients (docosahexanoic acid, vitamin C and E, and Bifidobacterium lactis strain BB-12®, included in an engineered diet) can act synergistically to reduce inflammation given by high level of air pollution. Beside the role of docosahexanoic acid, vitamins C and E on airways inflammatory disease, no study examined the effect of the supplementation of this probiotic strain in pathological conditions caused by air pollution so far. Herein we used a well-established in vivo model for the study of pollution effects, which consists in female BALB/c mice receiving by pharyngeal aspiration either a sham or a particulate matter with diameter <2.5 µm (PM 2.5) containing aerosol. Before treatment, mice were fed either a chow or a supplemented diet. By performing histological analyses and gene expression profiles on lung sections and serum measurement of the cytokine interleukin 10, we found that a specific combination of all the aforementioned nutrients rather than nutrients alone had a synergistic protective effect against PM2.5-induced inflammation. In conclusion, our study support that a supplemental nutritional intervention based on a combination of the probiotic B. lactis BB-12, the anti-oxidant vitamin C and E, and the anti-inflammatory docosahexanoic acid represents a rational option for alleviating air pollution-related lung inflammation.

Transgenic mice with dominant negative PKC-theta in skeletal muscle: a new model of insulin resistance and obesity.

Protein kinase C theta (PKC-theta) is the PKC isoform predominantly expressed in skeletal muscle, and it is supposed to mediate many signals necessary for muscle histogenesis and homeostasis, such as TGFbeta, nerve-dependent signals and insulin. To study the role of PKC-theta in these mechanisms we generated transgenic mice expressing a "kinase dead" mutant form of PKC-theta (PKC-thetaK/R), working as "dominant negative," specifically in skeletal muscle. These mice are viable and fertile, however, by the 6-7 months of age, they gain weight, mainly due to visceral fat deposition. Before the onset of obesity (4 months of age), they already show increased fasting and fed insulin levels and reduced insulin-sensitivity, as measured by ipITT, but normal glucose tolerance, as measured by ipGTT. After the 6-7 months of age, transgenic mice develop hyperinsulinemia in the fasting and fed state. The ipGTT revealed in the transgenic mice both hyperglycemia and hyperinsulinemia. At the molecular level, impaired activation of the IR/IRS/PI3K pathway and a significant decrease both in the levels and in insulin-stimulated activation of the serine/threonine kinase Akt were observed. Taken together these data demonstrate that over-expression of dominant negative PKC-theta in skeletal muscle causes obesity associated to insulin resistance, as demonstrated by defective receptor and post-receptorial activation of signaling cascade.

Engineering of a mouse for the in vivo profiling of estrogen receptor activity.

In addition to their well known control of reproductive functions, estrogens modulate important physiological processes. The identification of compounds with tissue-selective activity will lead to new drugs mimicking the beneficial effects of estrogen on the prevention of osteoporosis and cardiovascular or neurodegenerative diseases, while avoiding its detrimental proliferative effects. As an innovative model for the in vivo identification of new selective estrogen receptor modulators (SERMs), we engineered a mouse genome to express a luciferase reporter gene ubiquitously. The constructs for transgenesis consist of the reporter gene driven by a dimerized estrogen-responsive element (ERE) and a minimal promoter. Insulator sequences, either matrix attachment region (MAR) or beta-globin hypersensitive site 4 (HS4), flank the construct to achieve a generalized, hormoneresponsive luciferase expression. In the mouse we generated, the reporter expression is detectable in all 26 tissues examined, but is induced by 17beta-estradiol (E2) only in 15 of them, all expressing estrogen receptors (ERs). Immunohistochemical studies show that in the mouse uterus, luciferase and ERs colocalize. In primary cultures of bone marrow cells explanted from the transgenic mice and in vivo, luciferase activity accumulates with increasing E(2) concentration. E2 activity is blocked by the ER full antagonist ICI 182,780. Tamoxifen shows partial agonist activity in liver and bone when administered to the animals. In the mouse system here illustrated, by biochemical, immunohistochemical, and pharmacological criteria, luciferase content reflects ER transcriptional activity and thus represents a novel system for the study of ER dynamics during physiological fluctuations of estrogen and for the identification of SERMs or endocrine disruptors.

Constitutive activation of NF-kappaB and T-cell leukemia/lymphoma in Notch3 transgenic mice.

The multiplicity of Notch receptors raises the question of the contribution of specific isoforms to T-cell development. Notch3 is expressed in CD4(-)8(-) thymocytes and is down-regulated across the CD4(-)8(-) to CD4(+)8(+) transition, controlled by pre-T-cell receptor signaling. To determine the effects of Notch3 on thymocyte development, transgenic mice were generated, expressing lck promoter-driven intracellular Notch3. Thymuses of young transgenics showed an increased number of thymocytes, particularly late CD4(-)8(-) cells, a failure to down-regulate CD25 in post-CD4(-)8(-) subsets and sustained activity of NF-kappaB. Subsequently, aggressive multicentric T-cell lymphomas developed with high penetrance. Tumors sustained characteristics of immature thymocytes, including expression of CD25, pTalpha and activated NF-kappaB via IKKalpha-dependent degradation of IkappaBalpha and enhancement of NF-kappaB-dependent anti-apoptotic and proliferative pathways. Together, these data identify activated Notch3 as a link between signals leading to NF-kappaB activation and T-cell tumorigenesis. The phenotypes of pre-malignant thymocytes and of lymphomas indicate a novel and particular role for Notch3 in co-ordinating growth and differentiation of thymocytes, across the pre-T/T cell transition, consistent with the normal expression pattern of Notch3.