Modulatory role of mTOR in trophoblast adaptive response in the early stage of placentation in sheep.

In brief: Fibroblast growth factor-2 (FGF2) is essential for early placenta development in sheep. This study shows that the mechanistic target of rapamycin is the key modulator of trophoblast adaptive response under FGF2 modulation. Abstract: During the early stage of placentation in sheep, normal conceptus development is affected by trophoblast cell functionality, whose dysregulation results in early pregnancy loss. Trophoblast metabolism is supported mainly by histotrophic factors, including fibroblast growth factor-2 (FGF2), which are involved in cell differentiation and function through the modulation of specific cellular mechanisms. The mechanistic target of rapamycin (mTOR) is known as a cellular 'nutrient sensor', but its downstream regulation remains poorly understood. The hypothesis was that during trophoblast development, the FGF2 effect is mediated by mTOR signalling pathway modulation. Primary trophoblast cells from 21-day-old sheep placenta were characterised and subjected to FGF2 and rapamycin treatment to study the effects on cell functionality and gene and protein expression profiles. The model showed mainly mononuclear cells with epithelial cell-like growth and placental morphological properties, expressing typical trophoblast markers. FGF2 promoted cell proliferation and migration under normal culture conditions, whereas mTOR inhibition reversed this effect. When the mTOR signalling pathway was activated, FGF2 failed to influence invasion activity. mTOR inhibition significantly reduced cell motility, but FGF2 supplementation restored motility even when mTOR was inhibited. Interestingly, mTOR inhibition influenced endocrine trophoblast marker regulation. Although FGF2 supplementation did not affect ovine placenta lactogen expression, as observed in the control, interferon-tau was drastically reduced. This study provides new insights into the mechanism underlying mTOR inhibitory effects on trophoblast cell functionality. In addition, as mTOR is involved in the expression of hormonal trophoblast markers, it may play a crucial role in early placenta growth and fetal-maternal crosstalk.

Whole transcriptome analysis of bovine mammary progenitor cells by P-Cadherin enrichment as a marker in the mammary cell hierarchy.

Adult bovine mammary stem cells possess the ability to regenerate in vivo clonal outgrowths that mimic functional alveoli. Commonly available techniques that involve immunophenotype-based cell sorting yield cell fractions that are moderately enriched, far from being highly purified. Primary bovine mammary epithelial cells segregated in four different populations according to the expression of P-Cadherin and CD49f. Sorted cells from each fraction were tested for the presence of lineage-restricted progenitors and stem cells. Only cells from the CD49fhigh/P-Cadherinneg subpopulation were able to give rise to both luminal- and myoepithelial-restricted colonies in vitro and generate organized outgrowths in vivo, which are hallmarks of stem cell activity. After whole transcriptome analysis, we found gene clusters to be differentially enriched that relate to cell-to-cell communication, metabolic processes, proliferation, migration and morphogenesis. When we analyzed only the genes that were differentially expressed in the stem cell enriched fraction, clusters of downregulated genes were related to proliferation, while among the upregulated expression, cluster of genes related to cell adhesion, migration and cytoskeleton organization were observed. Our results show that P-Cadherin separates mammary subpopulations differentially in progenitor cells or mammary stem cells. Further we provide a comprehensive observation of the gene expression differences among these cell populations which reinforces the assumption that bovine mammary stem cells are typically quiescent.

Correlation between estrogen plasma level and miRNAs in muscle of Piedmontese cattle.

A loss-of-function mutation of the myostatin gene has a very high prevalence in the Piedmontese cattle breed. The effect of such mutation is a double-muscle phenotype because of hypertrophy. However, differences in muscle mass development can still be detected in individuals of this breed. Such differences must be generated by other factors controlling skeletal muscle development. MicroRNAs are short noncoding RNA molecules that modulate gene expression at a post-transcriptional level. MicroRNAs have been demonstrated to be involved in skeletal muscle development, and some of them are controlled by steroid hormone signaling. Data on estrogen signaling are lacking, whereas more studies have been carried out on the effect of androgens. We aimed at identifying putative estrogen responsive miRNAs that might be involved in skeletal muscle development. At a slaughterhouse, we collected muscle samples from longissimus dorsi and blood samples. Blood 17ß-estradiol concentration was assessed, and RNA was extracted from muscle samples. The animals we sampled were divided into groups according to estrogen blood concentration, and through qPCR expression, levels of 7 muscle-related miRNAs were evaluated. We found that miR-26b (P < 0.01), miR-27a-5p (P < 0.05), miR-27b (P < 0.05), and miR-199a-3p (P < 0.01) were differentially expressed among experimental groups. Expression levels of miR-26b were reduced approximately 50% in samples with a low blood estrogen concentrations, and the other miRNAs showed a tendency to increase their expression levels when blood estrogen levels were higher. The variations of the circulating concentrations of estrogen in Piedmontese cattle might influence muscle mass development through miRNAs and thus contribute to individual variability in a breed with a high prevalence of a myostatin point mutation.

Inhibition of a Descending Prefrontal Circuit Prevents Ketamine-Induced Stress Resilience in Females.

Stress is a potent etiological factor in the onset of major depressive disorder and posttraumatic stress disorder (PTSD). Therefore, significant efforts have been made to identify factors that produce resilience to the outcomes of a later stressor, in hopes of preventing untoward clinical outcomes. The NMDA receptor antagonist ketamine has recently emerged as a prophylactic capable of preventing neurochemical and behavioral outcomes of a future stressor. Despite promising results of preclinical studies performed in male rats, the effects of proactive ketamine in female rats remains unknown. This is alarming given that stress-related disorders affect females at nearly twice the rate of males. Here we explore the prophylactic effects of ketamine on stress-induced anxiety-like behavior and the neural circuit-level processes that mediate these effects in female rats. Ketamine given one week prior to an uncontrollable stressor (inescapable tailshock; IS) reduced typical stress-induced activation of the serotonergic (5-HT) dorsal raphe nucleus (DRN) and eliminated DRN-dependent juvenile social exploration (JSE) deficits 24 h after the stressor. Proactive ketamine altered prelimbic cortex (PL) neural ensembles so that a later experience with IS now activated these cells, which it ordinarily would not. Ketamine acutely activated a PL to DRN (PL-DRN) circuit and inhibition of this circuit with Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) at the time of IS one week later prevented stress prophylaxis, suggesting that persistent changes in PL-DRN circuit activity are responsible, at least in part, for mediating long-term effects associated with ketamine.

Mechanisms of modulation of the Egr gene family in mammary epithelial cells of different species.

In the adult female, within the estrous cycle, the mammary gland undergoes multiple rounds of growth, with increased cellular proliferation, and involution, with increased apoptosis. The increase in proliferation is elicited by endocrine (Estrogen, Progesterone), as well as locally produced (epidermal growth factor, insulin-like growth factor, etc) growth factors. Among the genes that are modulated during cellular proliferation, immediate early genes play a fundamental role, being rapidly upregulated and then downregulated within the G0/G1 phase of the cell cycle, allowing the progression to the subsequent phases. Egrs (1-4) are immediate early genes that encode for transcription factors that promote, within different cell types and depending on the strength and duration of the stimuli, several different responses like mitogenesis, differentiation, apoptosis or even anti-apoptosis. In this work we have studied the mechanisms of modulation of the Egr family, in mammary epithelial cells of different origin (bovine, canine, feline, murine). Following stimulation with growth medium, Egr mRNA expression showed a strong upregulation reaching a peak at 45-60min, that rapidly declined. Among several cytokines, particularly important for mammary morphogenesis, that we have tested (EGF, IGF-I, insulin, estrogen, progesterone), only EGF upregulated Egrs to levels close to those elicited by growth medium. In order to understand how the Egr transcription factors were regulated, we have inhibited Erk 1/2 and PI3K, molecules that drive two major intracellular signaling pathways. Inhibition of the Erk 1/2 pathway totally abolished Egr upregulation mediated by growth medium or EGF. On the other hand, the PI3K-Akt pathway played a minor role on Egr levels, with a strong inhibitory effect on cat GH2 cells only, that could be ascribed to reduced Erk phosphorylation following PI3K inhibition. Finally we showed that addition of growth medium also upregulated that the mammary luminal marker cytokeratin 18, but only in the murine NMuMG cell line. This is the first manuscript describing how the Egr transcription factors are expressed in mammary epithelial cells of domestic animals and which growth factors and signaling pathways modulate their expression.

Temporal correlation between differentiation factor expression and microRNAs in Holstein bovine skeletal muscle.

Satellite cells are adult stem cells located between the basal lamina and sarcolemma of muscle fibers. Under physiological conditions, satellite cells are quiescent, but they maintain a strong proliferative potential and propensity to differentiate, which underlies their critical role in muscle preservation and growth. MicroRNAs (miRNAs) play essential roles during animal development as well as in stem cell self-renewal and differentiation regulation. MiRNA-1, miRNA-133a and miRNA-206 are closely related muscle-specific miRNAs, and are thus defined myomiRNAs. MyomiRNAs are integrated into myogenic regulatory networks. Their expression is under the transcriptional and post-transcriptional control of myogenic factors and, in turn, they exhibit widespread control of muscle gene expression. Very little information is available about the regulation and behavior of satellite cells in large farm animals, in particular during satellite cell differentiation. Here, we study bovine satellite cells (BoSCs) undergoing a differentiation process and report the expression pattern of selected genes and miRNAs involved. Muscle samples of longissimus thoracis from Holstein adult male animals were selected for the collection of satellite cells. All satellite cell preparations demonstrated myotube differentiation. To characterize the dynamics of several transcription factors expressed in BoSCs, we performed real-time PCR on complementary DNA generated from the total RNA extracted from BoSCs cultivated in growth medium (GM) or in differentiation medium (DM) for 4 days. In the GM condition, BoSCs expressed the satellite cell lineage markers as well as transcripts for the myogenic regulatory factors. At the time of isolation from muscle, PAX7 was expressed in nearly 100% of BoSCs; however, its messenger RNA (mRNA) levels dramatically decreased between 3 and 6 days post isolation (P<0.01). MyoD mRNA levels increased during the 1st day of cultivation in DM (day 7; P<0.02), showing a gradual activation of the myogenic gene program. During the subsequent 4 days of culture in DM, several tested genes, including MRF4, MYOG, MEF2C, TMEM8C, DES and MYH1, showed increased expression (P<0.05), and these levels remained high throughout the culture period investigated. Meanwhile, the expression of genes involved in the differentiation process also miRNA-1, miRNA-133a and miRNA-206 were strongly up-regulated on the 1st day in DM (day 7; P<0.05). Analysis revealed highly significant correlations between myomiRNAs expression and MEF2C, MRF4, TMEM8C, DES and MYH1 gene expression (P<0.001). Knowledge about the transcriptional changes correlating with the growth and differentiation of skeletal muscle fibers could be helpful for developing strategies to improve production performance in livestock.

Overstocking dairy cows during the dry period affects dehydroepiandrosterone and cortisol secretion.

Stressful situations trigger several changes such as the secretion of cortisol and dehydroepiandrosterone (DHEA) from the adrenal cortex, in response to ACTH. The aim of this study was to verify whether overstocking during the dry period (from 21±3 d to the expected calving until calving) affects DHEA and cortisol secretion and behavior in Holstein Friesian cows. Twenty-eight cows were randomly divided into 2 groups (14 animals each), balanced for the number of lactations, body condition score, and expected date of calving. Cows in the far-off phase of the dry period (from 60 to 21 d before the expected calving date) were housed together in a bedded pack. Then, animals from 21±3 d before the expected calving until calving were housed in pens with the same size but under different crowding conditions due to the introduction of heifers (interference animals) into the pen. The control condition (CTR) had 2 animals per pen with 12.0m2 each, whereas the overstocked condition (OS) had 3 interference animals in the same pen with 4.8m2 for each animal. On d -30±3, -21±3, -15±3, -10±3, and -5±3 before and 10, 20, and 30 after calving, blood samples were collected from each cow for the determination of plasma DHEA and cortisol concentrations by RIA. Rumination time (min/d), activity (steps/h), lying time (min/d), and lying bouts (bouts/d) were individually recorded daily. In both groups, DHEA increased before calving and the concentration declined rapidly after parturition. Overstocking significantly increased DHEA concentration compared with the CTR group at d -10 (1.79±0.09 vs. 1.24±0.14 pmol/mL), whereas an increase of cortisol was observed at d -15 (3.64±0.52 vs. 1.64±0.46ng/mL). The OS group showed significantly higher activity (steps/h) compared with the CTR group. Daily lying bouts tended to be higher for the OS group compared with CTR group in the first week of treatment. The overall results of this study documented that overstocking during the dry period was associated with a short-term changes in DHEA and cortisol but these hormonal modifications did not influence cow behavior.

Differential expression of living mammary epithelial cell subpopulations in milk during lactation in dairy cows.

Epithelial cells are shed into milk during lactation, and although they generally reflect the cellular characteristics of terminally differentiated luminal cells, previously the detection of more primitive cells was described in human milk where a cell population of epithelial lineage was detected expressing markers typical of progenitor cells. In this investigation, we report the development of flow cytometry analysis to allow multiparametric assessment of mammary epithelial cells observed in milk. Cells collected from milk samples of 10 healthy dairy cows were directly analyzed for 6 different markers: CD45, CD49f, cytokeratin 14, cytokeratin 18, presence of nucleus, and cell viability. Milk samples were collected in 3 different periods of lactation: early lactation (EL=d 0-30), mid-lactation (ML=d 90-120), and late lactation (LL=210-250). Here we identify the differential expression of precursor or differentiated cell markers (or both) in mammary epithelial cells present in bovine milk. Myoepithelial cells, as indicated by cells staining positively for cytokeratin 14(+)/cytokeratin 18(-), were observed to increase from EL to LL with a high correlation with nuclear staining inferring potential proliferative activity. Furthermore, a significant increase in CD49f(+) and cytokeratin 14(+)/cytokeratin 18(+) positive cells was observed in LL. This assay is a sensitive approach for evaluating the variations in the frequency and features of living epithelial cells, whose reciprocal balance may be significant in understanding mammary gland cellular function throughout lactation. These observations suggest that mammary epithelial cell immunophenotypes could be investigated as biomarkers for mammary gland function in dairy cows.

DAT isn't all that: cocaine reward and reinforcement require Toll-like receptor 4 signaling.

The initial reinforcing properties of drugs of abuse, such as cocaine, are largely attributed to their ability to activate the mesolimbic dopamine system. Resulting increases in extracellular dopamine in the nucleus accumbens (NAc) are traditionally thought to result from cocaine's ability to block dopamine transporters (DATs). Here we demonstrate that cocaine also interacts with the immunosurveillance receptor complex, Toll-like receptor 4 (TLR4), on microglial cells to initiate central innate immune signaling. Disruption of cocaine signaling at TLR4 suppresses cocaine-induced extracellular dopamine in the NAc, as well as cocaine conditioned place preference and cocaine self-administration. These results provide a novel understanding of the neurobiological mechanisms underlying cocaine reward/reinforcement that includes a critical role for central immune signaling, and offer a new target for medication development for cocaine abuse treatment.

Bovine mammary stem cells: new perspective for dairy science.

Mammary stem cells provide opportunities for the cyclic remodelling of the bovine mammary gland. Therefore, understanding the character and regulation of mammary stem cells is important for increasing animal health and productivity. The exciting possibility that stem cell expansion can influence milk production is currently being investigated by several researchers. In fact, appropriate regulation of mammary stem cells could hopefully benefit milk yield, persistency of lactation, dry period management and tissue repair. Accordingly, we and others have attempted to characterize and regulate the function of bovine mammary stem cells. However, research on mammary stem cells requires tissue biopsies, which represents a limitation for the management of animal welfare. Interestingly, different studies recently reported the identification of putative mammary stem cells in human breast milk. The possible identification of primitive cell types within cow's milk may provide a non-invasive source of relevant mammary cells for a wide range of applications. In this review, we have summarized the main achievements in this field for dairy cow science and described the interesting perspectives open to manipulate milk persistency during lactation and to cope with oxidative stress during the transition period by regulating mammary stem cells.

Dynamic microglial alterations underlie stress-induced depressive-like behavior and suppressed neurogenesis.

The limited success in understanding the pathophysiology of major depression may result from excessive focus on the dysfunctioning of neurons, as compared with other types of brain cells. Therefore, we examined the role of dynamic alterations in microglia activation status in the development of chronic unpredictable stress (CUS)-induced depressive-like condition in rodents. We report that following an initial period (2-3 days) of stress-induced microglial proliferation and activation, some microglia underwent apoptosis, leading to reductions in their numbers within the hippocampus, but not in other brain regions, following 5 weeks of CUS exposure. At that time, microglia displayed reduced expression of activation markers as well as dystrophic morphology. Blockade of the initial stress-induced microglial activation by minocycline or by transgenic interleukin-1 receptor antagonist overexpression rescued the subsequent microglial apoptosis and decline, as well as the CUS-induced depressive-like behavior and suppressed neurogenesis. Similarly, the antidepressant drug imipramine blocked the initial stress-induced microglial activation as well as the CUS-induced microglial decline and depressive-like behavior. Treatment of CUS-exposed mice with either endotoxin, macrophage colony-stimulating factor or granulocyte-macrophage colony-stimulating factor, all of which stimulated hippocampal microglial proliferation, partially or completely reversed the depressive-like behavior and dramatically increased hippocampal neurogenesis, whereas treatment with imipramine or minocycline had minimal or no anti-depressive effects, respectively, in these mice. These findings provide direct causal evidence that disturbances in microglial functioning has an etiological role in chronic stress-induced depression, suggesting that microglia stimulators could serve as fast-acting anti-depressants in some forms of depressive and stress-related conditions.

Osteo-regenerative potential of ovarian granulosa cells: an in vitro and in vivo study.

Granulosa cells (GC) express stemness markers and can differentiate into cell types not present within the follicles. Given that follicles at different stages of development populate the ovary, we undertook this research in the pig model to identify the stage of follicle, growing or luteinizing, from which GC with the best regenerative potential can be retrieved. Growing follicles were isolated from prepubertal gilts 50 h after equine chorionic gonadotropin (eCG) (1,200 IU) administration. Luteinizing follicles were obtained from prepubertal gilts treated with eCG (1,200 IU) followed, 60 h later, by hCG (500 IU). The follicles were isolated 30 h after hCG. The GC isolated from growing (GGC) and from luteinizing (LGC) follicles were expanded in vitro for three passages and exposed to osteogenic medium to trigger differentiation. The GC incorporated in PLGA scaffolds were cultured in osteogenic medium for 2 wks and then implanted subcutaneously in the dorsal region of SCID mice to assess their osteogenic potential in vivo. In addition to the typical granulosa cells characteristics (inhibin, progesterone and estrogen production and FSH receptors), GGC and LGC showed a diffused expression of the stemness markers Sox2, Nanog and TERT immediately after isolation. Expansion caused in both cell types a rapid disappearance of granulosa cell characters while it did not modify stemness marker expression. Osteogenic medium induced a marked extracellular matrix mineralization and alkaline phosphatase activation in LGC, clearly detectable after two wks, while the process was much lighter in GGC, where it became evident after 3 wks. Osteocalcin and Runx2 expressions were upregulated and stemness markers downregulated by osteogenic medium. The GC loaded implants, retrieved 8 wks after transplantation, had viable GC surrounding the several nodules of calcifications recorded. Similar effects were induced by GGC and LGC while calcification nodules were not recorded when scaffolds without cells were implanted. These data confirm that GC, expanded in vitro undergo progressive de-differentiation retaining their plasticity and demonstrate that both GGC and LGC have osteogenic potential, luteinizing cells being more efficient. Transplanted in SCID mice, GC participate in new bone formation, thus confirming their therapeutic potential.

miRNAs highlights in stem and cancer cells.

MicroRNAs (miRNAs) are approximately 22 nucleotide endogenous RNA molecules which exert their functions by base pairing with messenger RNAs (mRNAs), thereby regulating protein-coding gene expression. In eukaryotic cells, miRNAs play important roles in regulating biological processes such as proliferation, differentiation, apoptosis, and stem cell self-renewal. miRNAs are encoded by the genome, and more than 1,000 human miRNAs have been identified so far. miRNAs are predicted to target -60% of human mRNAs and are expressed in all animal cells. Unique expression domains, targets, and gain- and loss-of-function phenotypes of particular miRNAs have important implications for directed to control differentiation of stem cell populations. Many cancers show variations in miRNA levels, and more specifically an overall downregulation, when compared to their normal counterparts. Therefore, miRNAs may be used as potential therapeutic agents to correct aberrant transcript levels found in the signaling pathways of cancer. This review examines the most recent acquisition on the role of miRNAs in regulating the cell cycle, with particular emphasis on their effects on cell proliferation and differentiation. The second part explores specifically the role of these factors in the physiological regulation of embryonic stem cells, of cellular reprogramming and their involvement in the activation of stem cells in adult tissues. In the third part, the article discusses some issues that relate to the role of miRNAs in the development of neoplastic diseases, focusing on aspects of the genetic and transcriptional alterations that determine the beginning and the development of tumor process, with emphasis on, looking to emphasize their involvement in the activation of adult cancer stem cells.

The scatter factor signaling pathways as therapeutic associated target in cancer treatment.

Receptor tyrosine kinases (RTKs) are key regulators of critical cellular processes such as proliferation, differentiation, neo-vascularization, and tissue repair. In addition to their importance in the regulation of normal physiology, aberrant expression of certain RTKs has also been associated to the development and progression of many types of cancer. c-Met and RON are two RTKs with closely related sequences, structural homology, and similar functional properties. Both these receptors, once activated by their respective ligands, the Hepatocyte Growth Factor/Scatter Factor (HGF/SF1) and the Macrophage Stimulating Protein/Scatter Factor 2 (MSP/SF2), can induce cell migration, invasion and proliferation. Soon after its discovery in the mid-1980s, c-Met attracted a great interest because of its role in modulating cell motility. Moreover, the causal role for c-Met activating mutations in human cancer propelled an intensive drug discovery effort throughout academic institutions and pharmaceutical companies. While c-Met is now a well-accepted target for anticancer drug design, less is known about the role of RON in cancer and less has been done to target this receptor. In this review we will discuss the biological relevance of c-Met and RON, their deregulation in human cancers and the progress, so far, in identifying c-Met and RON signaling inhibitors. Finally, we will focus on the development of therapeutic strategies and drug efficacy studies based on interfering the scatter factor signaling pathways.

Canine CYP2B11 metabolizes and is inhibited by anesthetic agents often co-administered in dogs.

Medetomidine is a well-established sedative and analgesic for dogs and cats. As a premedicant for anesthesia regimens that also include other agents, medetomidine can also provide a dose-sparing effect. While there are likely several reasons for the dose-sparing effect of medetomidine, the role of metabolic drug-drug interactions at the single enzyme level has not yet been examined. Using a panel of individually expressed canine cytochromes P450 cloned from beagle liver, this report demonstrates that medetomidine is an extremely potent CYP2B11 inhibitor (IC(50) < 10 nm) and that ketamine and midazolam are CYP2B11 substrates with high intrinsic clearances. These in vitro findings suggest that under some circumstances, medetomidine (i.e. 'perpetrator') may inhibit the metabolic clearance of some high metabolic clearance drugs (i.e. 'victims') with which it is commonly co-administered via the CYP2B11 pathway. However, as the dose-sparing effect of medetomidine premedication commonly results in anesthetic dose reduction, any increased plasma concentrations of victim drugs caused by medetomidine would still be lower than if no dose reduction had been made. Further studies are needed to characterize whether medetomidine possesses the potential to cause pharmacokinetic interactions. In conclusion, the ability of recombinant P450s to define canine-specific drug clearance pathways and P450 inhibitors should prove useful in identifying drug combinations that may require dose adjustments in dogs.

Positive effect of silymarin on cell growth and differentiation in bovine and murine mammary cells.

Silymarin, a naturally acknowledged hepatoprotector used in humans to treat liver diseases has been tested in murine (HC11) and bovine (BME-UV) mammary epithelial cell lines to evaluate a possible direct effect on cell growth and differentiation in mammary gland. Silymarin enhanced cell proliferation (p < 0.05) from 10 to 1000 ng/ml in association with growth factors, (up to 20%) or alone (up to 15%) versus controls. Furthermore, silymarin (100 ng/ml) was able to increase (p < 0.05) beta-casein gene expression alone or in association with prolactin (5 microg/ml). These effects may be related with protein kinase B (AKT) activation induced by silymarin treatment (p < 0.05) and/or by a dose-related inhibitory effect (p < 0.05) on caspase-3 activity related to a protective role in cell apoptosis. These data suggest that silymarin should be considered a candidate to support mammary gland activity during a lactogenetic state.

Pharmacokinetics and anti-inflammatory pharmacodynamics of prednisolone in cynomolgus monkey.

The purpose was to investigate whether the pharmacokinetics and pharmacodynamics of prednisolone in the non-human primate was an appropriate surrogate for man. After single intravenous doses of 0.03, 0.3, and 3 mg kg(-1), prednisolone demonstrated a dose-dependent clearance and volume of distribution. When corrected for concentration-dependent protein binding, the free clearance was linear at the tested dose levels. The protein binding-corrected volume of distribution was similar across doses. The serum half-life was estimated as being between 2 and 4 h. Prednisolone exhibits near complete inhibition of the cytokines TNF-alpha, IL-1beta, IL-6 and IL-8 with very similar IC50 estimates from 0.09 to 0.16 microg ml(-1) (from 0.24 to 0.44 microM). The monkey demonstrated a similar pharmacokinetics-pharmacodynamics profile of prednisolone when compared with man (from the literature).

Epidermal growth factor and hepatocyte growth factor receptors collaborate to induce multiple biological responses in bovine mammary epithelial cells.

The aim of this work was to explore whether epidermal growth factor (EGF) and hepatocyte growth factor (HGF) could increase the biological responses of a mammary epithelial cell line of bovine origin when added simultaneously. We also investigated a possible molecular mechanism underlying this cooperation. The development of mammary gland requires several circulating and locally produced hormones. Hepatocyte growth factor and its tyrosine kinase receptor, mesenchymal-epithelial transition factor (MET), are expressed and temporally regulated during mammary development and differentiation. Epidermal growth factor receptor and its ligands have also been implicated in the growth and morphogenesis of the mammary epithelium. Both EGF and HGF seem to exert a morphogenic program in this tissue; therefore, we hypothesized that these cytokines could act cooperatively in bovine mammary epithelial cells. We have already shown that the bovine BME-UV cell line, a nontumorigenic mammary epithelial line, expresses both MET and EGF receptor. Simultaneous treatment with HGF and EGF elicited an increase in proliferation, dispersion, degradation of extracellular matrix, and motility. Following EGF treatment, BME-UV mammary cells exhibited an increase in MET expression at both the mRNA and protein levels. Long-term treatment of BME-UV cells with HGF and EGF together increased the level of activation of the extracellular signal-regulated kinase 1/2 and protein kinase B signaling pathways when compared with HGF or EGF alone. These data outline a possible cooperative role of the EGF and HGF pathways and indicate that cross-talk between their respective receptors may modulate mammary gland development in the cow.

Effect of Echinacea augustifolia extract on cell viability and differentiation in mammary epithelial cells.

Echinacea spp. are popularly used as an herbal medicine or food supplement for enhancing the immune system and activating biological property in different tissues. In this study we show the biological effect of Echinacea augustifolia extract on cell viability and cell differentiation in mammary epithelial cell lines. These effects have been observed in two different cell line derived from mouse (HC11) and bovine (BME-UV). Echinacea extract enhanced cell liability from 100 to 1000 ng/ml in association with growth factors, epidermal growth factor (EGF) or insulin, but also without EGF (p<0.05) up to 37% vs. control. This effect may be modulated by MAPK and Akt activation that Echinacea extract treatment increased and/or by a reduction of caspase 3 activity, showed a dose-response decrease after Echinacea treatment. Finally Echinacea extract was able to increase (p<0.05) at 100 ng/ml beta-casein expression in association with PRL (5 microg/ml). These data demonstrate that Echinacea angustifolia extract can stimulate mammary epithelial cell physiology and may be considered a candidate to support mammary gland activity during a mammogenetic and lactogenetic state.