Microbial-immune cross-talk and regulation of the immune system

We are all born germ-free. Following birth we enter into a lifelong relationship with microbes residing on our body's surfaces. The lower intestine is home to the highest microbial density in our body, which is also the highest microbial density known on Earth (up to 10(12) /g of luminal contents). With our indigenous microbial cells outnumbering our human cells by an order of magnitude our body is more microbial than human. Numerous immune adaptations confine these microbes within the mucosa, enabling most of us to live in peaceful homeostasis with our intestinal symbionts. Intestinal epithelial cells not only form a physical barrier between the bacteria-laden lumen and the rest of the body but also function as multi-tasking immune cells that sense the prevailing microbial (apical) and immune (basolateral) milieus, instruct the underlying immune cells, and adapt functionally. In the constant effort to ensure intestinal homeostasis, the immune system becomes educated to respond appropriately and in turn immune status can shape the microbial consortia. Here we review how the dynamic immune-microbial dialogue underlies maturation and regulation of the immune system and discuss recent findings on the impact of diet on both microbial ecology and immune function.

Estradiol and insulin cross -talk in breast cancer

Approximately 33% of clinical breast carcinomas require estrogens to proliferate. Epidemiological data show that insulin resistance and diabetes mellitus is 2–3 times more prevalent in women with breast cancer than those with benign breast lesions, suggesting a clinical link between insulin and estradiol. Insulin and estradiol have a synergistic effect on the growth of MCF7 breast cancer cells, and long-term estradiol treatment upregulates the expression of the key insulin signaling protein IRS-1. The goal of this study was to further define the mechanism(s) of cross-talk between insulin and estradiol in regulating the growth of breast cancer. Using MCF7 cells, acute treatment with insulin or estradiol alone was found to stimulate two activities associated with growth: Erk MAP kinase and PI 3-kinase. However, combined acute treatment had an antagonistic effect on both activities. Acute estradiol treatment inhibited the insulin-stimulated tyrosine phosphorylation of IRS-1 while increasing its serine phosphorylation; the serine phosphorylation was attenuated by the PI 3-kinase inhibitor wortmannin. The acute antagonism observed with combined estradiol and insulin are not consistent with the long-term synergistic effect on growth. In contrast, chronic estradiol treatment enhanced the insulin-sensitivity of breast cancer cells as measured by increases in total cellular insulin-stimulated tyrosine phosphorylation of IRS-1 and activation of PI 3-kinase. Estradiol stimulation of gene transcription was found to require PI 3-kinase activity but not MAP kinase activity. Insulin alone had no effect on ER transcriptional activity, but chronic treatment in combination with estradiol resulted in synergism of ER transcription. The synergistic effect of insulin and estradiol on MCF7 cell growth was also found to require PI 3-kinase but not MAP kinase activity. Therefore, chronic estradiol treatment increases insulin stimulation of PI 3-kinase, and PI 3-kinase is required for estradiol stimulation of gene transcription alone and in combined synergy with insulin. These data demonstrate that PI 3-kinase is the locus for the cross-talk between insulin and estradiol which results in enhanced breast cancer growth with long-term exposure to both hormones. This may have important clinical implications for women with high risk for breast cancer and/or diabetes mellitus. ^

Molecular analysis of endo-β-mannanase genes upon seed imbibition suggest a cross-talk between radicle and micropylar endosperm during germination of Arabidopsis thaliana.

The endo-β-mannanase (MAN) family is represented in the Arabidopsis genome by eight members, all with canonical signal peptides and only half of them being expressed in germinating seeds. The transcripts of these genes were localized in the radicle and micropylar endosperm (ME) before radicle protrusion and this expression disappears as soon as the endosperm is broken by the emerging radicle tip. However, only three of these MAN genes, AtMAN5, AtMAN7 and especially AtMAN6 influence the germination time (t50) as assessed by the analysis of the corresponding knock-out lines. The data suggest a possible interaction between embryo and ME regarding the role of MAN during the Arabidopsis germination process.

Cross-talk response analysis on a piezoelectric

The study of the cross-talk and its effects in the performance of a matrix array of piezoelectric elements is an important issue. This corresponds to the study of the cross mode of vibration of each one of the piezoelectric elements that form the ultrasonic array. The aim is to detect and measure the cross-talk that is generated for the cross mode of vibration. In order to accomplish this task, an array of 2x3 elements was designed and developed. This was constructed using 8 MHz piezoelectric ceramics. A number of configurations have been experimented, considering the excitation of an increasing number of elements, in order to detect and measure the propagation of wave interference. Initial results show the way cross-talk interferes the beam generated by the array, this causing attenuation of the main beam and other negative effects.

Efecto del Cross-talk en la respuesta de un Transductor Ultrasónico tipo PZT

El presente trabajo de tesis investiga el efecto del fenómeno conocido como “Cross-talk” generado por el modo lateral de vibración, en la respuesta de un transductor ultrasónico formado por un arreglo de elementos piezoeléctricos tipo PZT (Zircanato Titanato de Plomo), la investigación se lleva a cabo desde el punto de vista de la naturaleza física de este efecto, así como de los parámetros asociados al mismo, así como un análisis del efecto del “Cross-talk” en la respuesta del transductor, formado por arreglos de elementos piezoeléctricos. Diversas investigaciones han abordado el fenómeno del “Cross-talk” y de sus efectos en la respuesta de los transductores, estos se han enfocado principalmente al modo espesor (thickness) de vibración. Sin embargo no ha habido un estudio a fondo para el estudio de este fenómeno en el modo lateral de vibración tema de interés de este trabajo de tesis. Este trabajo incluye simulaciones del fenómeno del “Cross-talk” mediante el método de los elementos finitos (MEF), así como la construcción de un transductor tipo matricial (arrray) de 2x3 elementos, en el que fueron realizadas las mediciones físicas del fenómeno. El trabajo abarca un estudio comparativo entre las simulaciones y las mediciones realizadas en el transductor, considerando que las cerámicas del transductor están montadas sobre diferentes materiales (backing) en donde la propagación de la energía emitida por las cerámicas piezoeléctricas provoca un mayor o menor grado de “Cross-talk” dependiendo de la velocidad en que se propaga dicha energía. Esta investigación también llevó a cabo el estudio del efecto del “Cross-talk” en el patrón de radiación que emite el arreglo de elementos piezoeléctricos, siendo este patrón de radiación un factor importante en la respuesta del transductor, motivo por el cual se realizó un análisis de cómo se ve afectado este patrón bajo la influencia del fenómeno del “Cross-talk”. Como ya se mencionó debido a la falta de un estudio a profundidad del fenómeno del “Cross-talk” en el modo lateral, la contribución del presente trabajo es importante ya que se enfoca al modo lateral de vibración de los elementos piezoeléctricos del arreglo. En particular se desarrollo una ecuación que permite cuantificar el fenómeno del “Cross-talk” y visualizar sus efectos en el arreglo. Derivando de este estudio se concluye que el fenómeno del “Cross-talk” generado por el modo lateral de vibración tiene un efecto significativo en la respuesta de los diferentes transductores matriciales considerados. ABSTRACT This thesis investigates the effect of the phenomenon known as crosstalk from the point of view of its physical nature and the elements that lead to the formation of this phenomenon to an analysis of how it may affect the performance of the ultrasonic transducer. This phenomenon occurs primarily in matrix arrays and this phenomenon is magnified by certain factors causing serious problems in the performance of a transducer. Researchers have addressed the phenomenon of crosstalk and their effects on the response of these transducers. They have mainly focused in the thickness vibration mode, and there has been no comprehensive study of this phenomenon in the lateral vibration mode, issue of interest of this thesis. This work includes simulations of the crosstalk phenomenon using the finite element method (FEM), and the construction of a matrix type transducer (array) of 2x3 elements, in which physical measurements were made. The work includes a comparative study between simulations and measurements in the transducer, whereas the ceramic transducer are mounted on different materials (backing) where the spread of the energy emitted by the piezoelectric ceramic causes a greater or lesser degree of crosstalk depending on the speed at which this energy spreads. This research also carried out the study of the effect of the crosstalk in the radiation pattern emitted by the piezoelectric array. The radiation pattern is an important factor in the response of the transducer that is why we conducted an analysis of how this pattern is affected under the influence of the crosstalk phenomenon. As mentioned before because of the lack of an in-depth study of the crosstalk phenomenon in the lateral vibration mode, the contribution of this work is important because it focuses in this vibration mode of the piezoelectric elements in the array. In particular, an equation was developed to quantify the crosstalk phenomenon and to see its effects in the array. Deriving from this study it is possible to conclude that the crosstalk phenomenon generated by the lateral vibration mode has a significant effect on the response of the different matrix transducers considered in this work.

Cross-talk between the insulin and angiotensin signaling systems.

Angiotensin II (AII), acting via its G-protein linked receptor, is an important regulator of cardiac, vascular, and renal function. Following injection of AII into rats, we find that there is also a rapid tyrosine phosphorylation of the major insulin receptor substrates 1 and 2 (IRS-1 and IRS-2) in the heart. This phenomenon appears to involve JAK2 tyrosine kinase, which associates with the AT1 receptor and IRS-1/IRS-2 after AII stimulation. AII-induced phosphorylation leads to binding of phosphatidylinositol 3-kinase (PI 3-kinase) to IRS-1 and IRS-2; however, in contrast to other ligands, AII injection results in an acute inhibition of both basal and insulin-stimulated PI 3-kinase activity. The latter occurs without any reduction in insulin receptor or IRS phosphorylation or in the interaction of the p85 and p110 subunits of PI 3-kinase with each other or with IRS-1/IRS-2. These effects of AII are inhibited by AT1 receptor antagonists. Thus, there is direct cross-talk between insulin and AII signaling pathways at the level of both tyrosine phosphorylation and PI 3-kinase activation. These interactions may play an important role in the association of insulin resistance, hypertension, and cardiovascular disease.

Cross talk between cell death and cell cycle progression: BCL-2 regulates NFAT-mediated activation.

BCL-2-deficient T cells demonstrate accelerated cell cycle progression and increased apoptosis following activation. Increasing the levels of BCL-2 retarded the G0-->S transition, sustained the levels of cyclin-dependent kinase inhibitor p27Kip1, and repressed postactivation death. Proximal signal transduction events and immediate early gene transcription were unaffected. However, the transcription and synthesis of interleukin 2 and other delayed early cytokines were markedly attenuated by BCL-2. In contrast, a cysteine protease inhibitor that also blocks apoptosis had no substantial affect upon cytokine production. InterleUkin 2 expression requires several transcription factors of which nuclear translocation of NFAT (nuclear factor of activated T cells) and NFAT-mediated transactivation were impaired by BCL-2. Thus, select genetic aberrations in the apoptotic pathway reveal a cell autonomous coregulation of activation.

Single cell Ca2+/cAMP cross-talk monitored by simultaneous Ca2+/cAMP fluorescence ratio imaging.

The spatial and temporal dynamics of two intracellular second messengers, cAMP and Ca2+, were simultaneously monitored in living cells by digital fluorescence ratio imaging using FlCRhR, a single-excitation dual-emission cAMP indicator, and fura-2, a dual-excitation single-emission Ca2+ probe. In single C6-2B glioma cells, isoproterenol- or forskolin-evoked cAMP accumulation (measured in vivo as an increased FlCRhR emission ratio) was reduced when cytosolic free Ca2+ concentration was elevated before, simultaneously with, or after cAMP activation. However, in REF-52 fibroblasts, Ca2+ neither prevented nor reduced forskolin-stimulated cAMP production. These results provide novel in vivo evidence for the Ca2+ modulation of the cAMP transduction pathway in C6-2B cells. The simultaneous microscopic measurement of cAMP and Ca2+ kinetics in single cells makes it now possible to study the regulatory interactions between these second messengers at the cellular and even the subcellular level.

Viral cross talk: intracellular inactivation of the hepatitis B virus during an unrelated viral infection of the liver.

Hepatitis B virus (HBV) infection is thought to be controlled by virus-specific cytotoxic T lymphocytes (CTL). We have recently shown that HBV-specific CTL can abolish HBV replication noncytopathically in the liver of transgenic mice by secreting tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) after antigen recognition. We now demonstrate that hepatocellular HBV replication is also abolished noncytopathically during lymphocytic choriomeningitis virus (LCMV) infection, and we show that this process is mediated by TNF-alpha and IFN-alpha/beta produced by LCMV-infected hepatic macrophages. These results confirm the ability of these inflammatory cytokines to abolish HBV replication; they elucidate the mechanism likely to be responsible for clearance of HBV in chronically infected patients who become superinfected by other hepatotropic viruses; they suggest that pharmacological activation of intrahepatic macrophages may have therapeutic value in chronic HBV infection; and they raise the possibility that conceptually similar events may be operative in other viral infections as well.

Protein kinase cross-talk: membrane targeting of the beta-adrenergic receptor kinase by protein kinase C.

The beta-adrenergic receptor kinase (betaARK) is the prototypical member of the family of cytosolic kinases that phosphorylate guanine nucleotide binding-protein-coupled receptors and thereby trigger uncoupling between receptors and guanine nucleotide binding proteins. Herein we show that this kinase is subject to phosphorylation and regulation by protein kinase C (PKC). In cell lines stably expressing alpha1B- adrenergic receptors, activation of these receptors by epinephrine resulted in an activation of cytosolic betaARK. Similar data were obtained in 293 cells transiently coexpressing alpha1B- adrenergic receptors and betaARK-1. Direct activation of PKC with phorbol esters in these cells caused not only an activation of cytosolic betaARK-1 but also a translocation of betaARK immunoreactivity from the cytosol to the membrane fraction. A PKC preparation purified from rat brain phospborylated purified recombinant betaARK-1 to a stoichiometry of 0.86 phosphate per betaARK-1. This phosphorylation resulted in an increased activity of betaARK-1 when membrane-bound rhodopsin served as its substrate but in no increase of its activity toward a soluble peptide substrate. The site of phosphorylation was mapped to the C terminus of betaARK-1. We conclude that PKC activates betaARK by enhancing its translocation to the plasma membrane.

Thyroid hormone (T3) inhibits ciprofibrate-induced transcription of genes encoding beta-oxidation enzymes: cross talk between peroxisome proliferator and T3 signaling pathways.

Peroxisome proliferators cause rapid and coordinated transcriptional activation of genes encoding peroxisomal beta-oxidation system enzymes by activating peroxisome proliferator-activated receptor (PPAR) isoform(s). Since the thyroid hormone (T3; 3,3',5-triiodothyronine) receptor (TR), another member of the nuclear hormone receptor superfamily, regulates a subset of fatty acid metabolism genes shared with PPAR, we examined the possibility of interplay between peroxisome proliferator and T3 signaling pathways. T3 inhibited ciprofibrate-induced luciferase activity as well as the endogenous peroxisomal beta-oxidation enzymes in transgenic mice carrying a 3.2-kb 5'-flanking region of the rat peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase gene fused to the coding region of luciferase. Transfection assays in hepatoma H4-II-E-C3 and CV-1 cells indicated that this inhibition is mediated by TR in a ligand-dependent fashion. Gel shift assays revealed that modulation of PPAR action by TR occurs through titration of limiting amounts of retinoid X receptor (RXR) required for PPAR activation. Increasing amounts of RXR partially reversed the inhibition in a reciprocal manner; PPAR also inhibited TR activation. Results with heterodimerization-deficient TR and PPAR mutants further confirmed that interaction between PPAR and TR signaling systems is indirect. These results suggest that a convergence of the peroxisome proliferator and T3 signaling pathways occurs through their common interaction with the heterodimeric partner RXR.

Nur77 regulates lipolysis in skeletal muscle cells - Evidence for cross-talk between the beta-adrenergic and an orphan nuclear hormone receptor pathway

Skeletal muscle is a major mass peripheral tissue that accounts for similar to 40% of total body weight and 50% of energy expenditure and is a primary site of glucose disposal and fatty acid oxidation. Consequently, muscle has a significant role in insulin sensitivity, obesity, and the blood-lipid profile. Excessive caloric intake is sensed by the brain and induces beta-adrenergic receptor (beta-AR)- mediated adaptive thermogenesis. beta-AR null mice develop severe obesity on a high fat diet. However, the target gene(s), target tissues(s), and molecular mechanism involved remain obscure. We observed that 30 - 60 min of beta-AR agonist ( isoprenaline) treatment of C2C12 skeletal muscle cells strikingly activated (> 100-fold) the expression of the mRNA encoding the nuclear hormone receptor, Nur77. In contrast, the expression of other nuclear receptors that regulate lipid and carbohydrate metabolism was not induced. Stable transfection of Nur77-specific small interfering RNAs (siNur77) into skeletal muscle cells repressed endogenous Nur77 mRNA expression. Moreover, we observed attenuation of gene and protein expression associated with the regulation of energy expenditure and lipid homeostasis, for example AMP-activated protein kinase gamma 3, UCP3, CD36,adiponectin receptor 2, GLUT4, and caveolin-3. Attenuation of Nur77 expression resulted in decreased lipolysis. Finally, in concordance with the cell culture model, injection and electrotransfer of siNur77 into mouse tibialis cranialis muscle resulted in the repression of UCP3 mRNA expression. This study demonstrates regulatory cross-talk between the nuclear hormone receptor and beta-AR signaling pathways. Moreover, it suggests Nur77 modulates the expression of genes that are key regulators of skeletal muscle lipid and energy homeostasis. In conclusion, we speculate that Nur77 agonists would stimulate lipolysis and increase energy expenditure in skeletal muscle and suggest selective activators of Nur77 may have therapeutic utility in the treatment of obesity.

Rev-erb beta regulates the expression of genes involved in lipid absorption in skeletal muscle cells - Evidence for cross-talk between orphan nuclear receptors and myokines

Rev-erbbeta is an orphan nuclear receptor that selectively blocks trans-activation mediated by the retinoic acid-related orphan receptor-alpha (RORalpha). RORalpha has been implicated in the regulation of high density lipoprotein cholesterol, lipid homeostasis, and inflammation. Rev-erbbeta and RORalpha are expressed in similar tissues, including skeletal muscle; however, the pathophysiological function of Rev-erbbeta has remained obscure. We hypothesize from the similar expression patterns, target genes, and overlapping cognate sequences of these nuclear receptors that Rev-erbbeta regulates lipid metabolism in skeletal muscle. This lean tissue accounts for > 30% of total body weight and 50% of energy expenditure. Moreover, this metabolically demanding tissue is a primary site of glucose disposal, fatty acid oxidation, and cholesterol efflux. Consequently, muscle has a significant role in insulin sensitivity, obesity, and the blood-lipid profile. We utilize ectopic expression in skeletal muscle cells to understand the regulatory role of Rev-erbbeta in this major mass peripheral tissue. Exogenous expression of a dominant negative version of mouse Rev-erbbeta decreases the expression of many genes involved in fatty acid/lipid absorption (including Cd36, and Fabp-3 and -4). Interestingly, we observed a robust induction (> 15-fold) in mRNA expression of interleukin-6, an exercise-induced myokine that regulates energy expenditure and inflammation. Furthermore, we observed the dramatic repression (> 20- fold) of myostatin mRNA, another myokine that is a negative regulator of muscle hypertrophy and hyperplasia that impacts on body fat accumulation. This study implicates Rev-erbbeta in the control of lipid and energy homoeostasis in skeletal muscle. In conclusion, we speculate that selective modulators of Rev-erbbeta may have therapeutic utility in the treatment of dyslipidemia and regulation of muscle growth.

Cross-talk towards the response regulator NtrC controlling nitrogen metabolism in Rhodobacter capsulatus

Rhodobacter capsulatus NtrB/NtrC two-component regulatory system controls expression of genes involved in nitrogen metabolism including urease and nitrogen fixation genes. The ntrY-ntrX genes, which are located immediately downstream of the nifR3-ntrB-ntrC operon, code for a two-component system of unknown function. Transcription of ntrY starts within the ntrC-ntrY intergenic region as shown by primer extension analysis, but maximal transcription requires, in addition, the promoter of the nifR3-ntrB-ntrC operon. While ntrB and ntrY single mutant strains were able to grow with either urea or N-2 as sole nitrogen source, a ntrB/ntrY double mutant (like a ntrC-deficient strain) was no longer able to use urea or N-2. These findings suggest that the histidine kinases NtrB and NtrY can substitute for each other as phosphodonors towards the response regulator NtrC.