Increased dietary protein intake rapidly (3 h) decreases hepatic malic enzyme and increases hepatic histidase mRNA expression in broiler chicks. A series of experiments was conducted to determine the role that glucagon or a specific mixture of dietary amino acids might have in regulating the rapid changes in mRNA expression of these enzymes, when dietary protein intake is increased. Three hours after the injection of glucagon (240 microg/kg of BW) into the brachial vein of broiler chicks, hepatic malic enzyme mRNA expression was significantly lower and hepatic histidase mRNA expression was significantly greater than the level detected in saline-injected chicks. In addition, broiler chicks fed a high (40 g/ 100 g of diet) protein diet had significantly higher plasma glucagon levels at 1 and 3 h after initial access to this diet than broiler chicks fed a basal (22 g/100 g of diet) protein diet. The plasma glucagon concentration, however, was not different between the chicks fed the 2 dietary protein levels at 2 h after the initial access to the 2 diets. When a mixture of indispensable or dispensable amino acids was added to the basal diet to equal the concentrations of the individual indispensable or dispensable amino acids in the high protein diet, hepatic mRNA expression of malic enzyme and histidase were intermediate to the expression found in chicks fed the basal and high protein diet. The results indicate that glucagon may mediate the changes in the mRNA expression of malic enzyme and histidase in response to dietary protein intake and that total amino acid intake rather than the ingestion of specific amino acids regulates the mRNA expression of malic enzyme and histidase in chicks.

The protective mucus layer covers the entire surface of the gastrointestinal tract. The mucus layer also acts as a medium for molecule transport between the luminal contents and the enterocytes; therefore it has a major role in nutrient absorption. The main mucus layer component, mucin glycoproteins, is produced by mucous-secreting goblet cells. In chicken small intestine, functional development of goblet cells and enterocytes occurs in the late embryonic and immediate posthatch period. Presence of the nutrient is crucial for mucosal development. Feed deprivation immediately after hatch caused delayed mucosa development and perturbed mucin dynamics. Recent studies showed the intraamnionic nutrient supply (in-ovo feeding; IOF) accelerated mucosa functional development. In this study, the effect of IOF on the mucin mRNA expression and mucin content in the goblet cells was studied. The feeding solution containing carbohydrates was administered to the amnionic fluid of the Cobb embryos at d 17.5 of incubation. Samples from the jejunum were taken at d 17 of incubation (before IOF), and then 10 embryos from each group were sampled at 19 d of incubation, at hatch, and at d 3 posthatch. Following IOF, villus surface area increased at day of hatch and 3 d posthatch by 27 and 21%, respectively. In addition, the proportion of goblet cells containing acidic mucin increased 36 h after injection by 50% compared with the controls. The mucin mRNA expression increased gradually from d 17 of incubation to 3 d posthatch. Enhanced expression of the mucin mRNA was found at the day of hatch in chicks that received carbohydrate solution into the amnionic fluid in comparison with the control group. The results showed that providing the carbohydrates as an energy source to the late-term embryo had a trophic effect on the small intestine and enhanced goblet cell development.

We evaluated the effects of celecoxib treatment on tumor-infiltrating lymphocyte (TIL) subsets [CD3(+), CD4(+),CD8(+), CD25(+), and T cell receptor (TCR)-zeta-expressing cells] and tryptase-positive mast cells in cervical tumors. Circulating levels of cytokines [interleukin (IL)-1beta, IL-10, tumor necrosis factor-alpha, IL-6, and IL-12] and angiogenesis-modulating factors (vascular endothelial growth factor and endostatin) have also been analyzed.

This study describes the development of a rapid and practical real-time RT-PCR method to quantify ribonucleotide reductase M2 (RRM2) mRNA in tumor and peripheral white blood cells (WBCs) from patients treated with GTI-2040, an antisense drug currently in clinical trials. In order to assess target down-regulation by GTI-2040, RRM2 mRNA expression levels were analyzed in pre- and post-treatment samples from a phase II clinical trial of GTI-2040 combined with capecitabine in patients with metastatic breast cancer. Target gene RRM2 mRNA levels were evaluated using quantitative RT-PCR method: real-time PCR (TaqMan) with fluorescein labeled probes on an ABI 7900HT instrument, with additional post-processing of the data to adjust for differences in total RNA in-put across the samples. Data are presented from a patient for whom both biopsy and PBMC samples were available, demonstrating applicability of this reproducible, highly sensitive real-time RT-PCR method for the detection and quantification of mRNAs for RRM2 in human WBC and tissue samples. By providing quantitative measurement of changes in target gene expression, this method may provide an opportunity to determine the correlation between target response to GTI-2040 antisense and clinical response in patients. Furthermore this assay may assess whether WBC samples are an appropriate surrogate tissue for approximating target down-regulation in the tumor.

Glucocorticoid (GC) therapy is recognized to be effective for the treatment of recurrent airway obstruction (RAO) in horses. Anti-inflammatory properties of GC are thought to be mediated by suppression of inflammatory gene expression via inhibition of transcription factors such as nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1). The purpose of this study was to evaluate the effect of low-dose inhaled beclomethasone dipropionate and injectable dexamethasone 21-isonicotinate on clinical signs, pulmonary function, airway cytology, and activity of NF-kappaB and AP-1 in bronchial cells of RAO-affected horses. Seven horses with RAO were exposed to moldy hay until they developed airway obstruction on 3 separate occasions. In a crossover design, they were then treated with a placebo (injection on day 1), inhaled beclomethasone (500 microg q12h for 10 days), or dexamethasone (0.06 mg/kg, IM on day 1) and monitored for 10 days. Pulmonary function, bronchoalveolar lavage fluid cytology, and NF-kappaB and AP-1 activity in bronchial brushing cells were measured before (day 1) and after treatment (day 10). Treatment with beclomethasone resulted in significantly improved pulmonary function of RAO-affected horses compared with placebo and dexamethasone treatments. However, none of the treatments had an effect on bronchoalveolar lavage fluid cytology or NF-kappaB and AP-1 activity. These findings reveal that, in a model of severe RAO, the benefits of low-dose inhaled beclomethasone on pulmonary function are not accompanied by a decrease in airway inflammatory cells or a suppression of transcription factors NF-kappaB and AP-1 DNA-binding activity.

To determine whether endometrial expression of endothelial nitric oxide synthase (eNOS) or inducible NOS (iNOS) protein in women with endometriosis-associated infertility is different from that in the fertile controls, whether GnRH agonist (GnRH-a) regulates the endometrial expression of NOS in women with endometriosis-associated infertility, and whether there is a correlation between serum E2 or P levels, and the endometrial expression of eNOS or iNOS.

To evaluate the effects of recombinant human luteinizing hormone (rhLH) supplementation on ovarian stimulation and implantation rate in down-regulated women of advanced reproductive age.

The present study investigated the effects of injected darbepoetin [novel erythropoietin stimulating protein (NESP)] on the density of three erythrocyte membrane transport proteins: the lactate-H+ cotransporter (monocarboxylate transporter 1), the chloride/bicarbonate exchanger 1 (anion exchanger 1), and the water channel aquaporin 1. Thirteen subjects were injected with NESP once a week for 4 wk. Blood samples were obtained before, during, and after the injection period, and the erythrocyte transport proteins were determined by Western blotting. The NESP injections induced a transient increase in hematocrit, red cell volume, and reticulocyte fraction. The density of aquaporin 1 protein was higher (maximal increase +59%) (P < 0.01) during the injection period compared with the preinjection value and lower (P < 0.01) after the injection period. The density of anion exchanger 1 protein was higher (maximal increase +15%) (P < 0.05) during the injection period compared with the preinjection value and tended (P = 0.06) to be lower after the injection period than before the injection period. The density of the erythrocyte monocarboxylate transporter 1 protein was higher (maximal increase +43%) (P < 0.05) during the injection period than in the preinjection period. Age separation experiments using self-creating Percoll gradients demonstrated a higher density of membrane transport proteins in young red blood cells. These data suggest that the NESP-induced increase in membrane transport proteins is caused by a higher fraction of newly formed erythrocytes (and reticulocytes), which have a higher density of membrane transport proteins. However, increased incorporation of membrane proteins during erythrocyte formation may also be involved. We suggest that NESP improves the quality of erythrocyte membrane transport through these mechanisms.

The majority of epithelial ovarian carcinomas are of serous subtype, with most women presenting at an advanced stage. Approximately 70% respond to initial chemotherapy but eventually relapse. We aimed to find markers of treatment response that might be suitable for routine use, using the gene expression profile of tumor tissue. Thirty one women with histologically-confirmed late-stage serous ovarian cancer were classified into 3 groups based on response to treatment (nonresponders, responders with relapse less than 12 months and responders with no relapse within 12 months). Gene expression profiles of these specimens were analyzed with respect to treatment response and survival (minimum 36 months follow-up). Patients' clinical features did not correlate with prognosis, or with specific gene expression patterns of their tumors. However women who did not respond to treatment could be distinguished from those who responded with no relapse within 12 months based on 34 gene transcripts (p < 0.02). Poor prognosis was associated with high expression of inhibitor of differentiation-2 (ID2) (p = 0.001). High expression of decorin (DCN) and ID2 together was strongly associated with reduced survival (p = 0.003), with an estimated 7-fold increased risk of dying (95% CI 1.9-29.6; 14 months survival) compared with low expression (44 months). Immunohistochemical analysis revealed both nuclear and cytoplasmic distribution of ID2 in ovarian tumors. High percentage of nuclear staining was associated with poor survival, although not statistically significantly. In conclusion, elevated expression of ID2 and DCN was significantly associated with poor prognosis in a homogeneous group of ovarian cancer patients for whom survival could not be predicted from clinical factors.

To study the apoptosis induced by preoperative oral 5'-DFUR administration in gastric adenocarcinoma and its mechanism of action.

IL-6 mediates many aspects of the exercise-induced acute-phase response, including upregulation of antioxidant defenses. Moreover, IL-6 synthesis is regulated in part by oxidative stress. This investigation tested the hypothesis that an IL-6-mediated acute-phase response after exercise provides negative-feedback protection against exercise-induced oxidative stress. Healthy young (n = 16, 26.4 +/- 1.8 yr) and older men (n = 16, 71.1 +/- 2.0 yr) ran downhill for 45 min at 75% maximal oxygen consumption before and after a 12-wk period of supplementation with vitamin E (1,000 IU/day) or placebo. Circulating IL-6 and soluble IL-6 receptors, peripheral mononuclear cell production of IL-6, and IL-6 transcripts in muscle were measured before and within a 72-h time window after each acute exercise bout. At all time points plasma IL-6, IL-6 bioavailability, and C-reactive protein were higher in the older men; yet in response to exercise, young and older subjects experienced similar increases in these factors. Although the magnitude of postexercise changes in acute-phase variables was independent of age, correlations among plasma, mononuclear cell, and muscle IL-6 and oxidative stress were evident only in young men (R2 = 0.64, 0.35, and 0.33, respectively). These changes in circulating IL-6 were closely associated with a prooxidant state (R2 = 0.47), whereas muscle IL-6 mRNA correlated with an antioxidant state (R2 = 0.65). Supplementation with vitamin E did not affect exercise-induced responses or differences between the young and old men in a consistent manner. Therefore, oxidative stress is linked to the acute-phase response after exercise in young men, but not in older men who had elevated acute-phase reactants, suggesting that further research is warranted to determine the basis for these differences.

L-asparaginase (L-Asp) is an effective drug for treatment of children with acute lymphoblastic leukemia (ALL). The effectiveness is generally thought to result from a rapid depletion of asparagine in serum and cells. Asparagine synthetase (AS) opposes the action of L-Asp by resynthesis of asparagine. In vitro, resistance to L-Asp has been associated with up-regulation of AS mRNA expression. We monitored AS mRNA levels in leukemic cells before and during 5 days after intravenous administration of 1000 IU/m(2) pegylated L-asparaginase (PEG-Asp) in a therapeutic window in children with ALL at initial diagnosis. Within 24 hours, AS mRNA levels increased by 3.5-fold and remained stable in the following 4 days. Baseline and L-Asp-induced expression levels of AS did not differ between clinically good, intermediate, and poor responders to PEG-Asp. No significant difference of AS mRNA up-regulation was found between precursor B- and T-ALL or between hyperdiploids, TEL/AML1 rearranged ALL or absence of genetic abnormalities. In 3 of 12 patients with T-ALL even a slight down-regulation of AS mRNA expression upon L-Asp exposure was found. In conclusion, although L-Asp exposure induces the expression of AS mRNA, the up-regulated gene expression does not correlate with an early clinical poor response to this drug in children with ALL.

A total of 216 Brown Dwarf laying hens (1.62 +/- 0.06 kg BW and 60 wk old) were fed 1 of 3 corn-soybean meal-based diets containing 0, 2.5, or 5.0% conjugated linoleic aicd (CLA) to explore its effects on the fatty acid composition of egg yolk, plasma, and liver as well as hepatic stearoyl-coenzyme A desaturase-1 (SCD-1) activity and its mRNA gene expression. Four hens were placed in wired-floored cages (45 x 40 x 45 cm) and 3 cages were grouped as 1 replicate, resulting in 6 replicates per treatment. The experimental diets were fed for 54 d, and then eggs were collected to determine the fatty acid composition of egg yolk. Four eggs were randomly selected from the total day's production for each replicate, and the contents were pooled prior to analysis. On d 56, one randomly chosen hen from each replicate (6 hens per replicate and a total of 18 hens) was bled via heart puncture and then killed in order to collect liver samples to measure the fatty acid profile of plasma and liver tissue as well as hepatic SCD-1 activity and its mRNA abundance. Dietary supplementation of CLA resulted in a significant deposition of CLA in egg yolk, plasma, and liver lipids (P < 0.01). As the dietary level of CLA increased, the concentration of saturated fatty acids in egg yolk, plasma, and liver also increased (P < 0.05). However, the concentration of monounsaturated fatty acids in these same tissues decreased (P < 0.01). Compared with the control, the activity of SCD-1 was reduced by feeding 2.5% CLA (P < 0.05) without a change in SCD-1 mRNA gene expression. However, feeding 5% CLA reduced both SCD-1 activity and mRNA abundance (P < 0.05). These results indicate that the conversion of saturated to monounsaturated fatty acids in egg yolk, plasma, and liver might be modulated directly at hepatic mRNA gene expression levels, or may be indirectly regulated at the downstream post-transcriptional levels.

This phase II trial of pemetrexed explored potential correlations between treatment outcome (antitumor activity) and molecular target expression.

In addition to lipid lowering effects, statins appear to have pleiotropic immunomodulatory properties. As they particularly affect monocyte functions, we tested the influence of statin treatment on the monocyte activating toll-like receptors (TLR) 4 and 2 in response to lipopolysaccharides (LPS) in vivo. In this double-blind, placebo-controlled study, 20 healthy, male subjects were randomized to receive either simvastatin (80 mg/day) or placebo for 4 days before intravenous LPS administration (20 IU/kg). Simvastatin did not influence the increase in TLR transcripts after LPS administration measured in mRNA isolated from whole blood by quantitative RT-PCR. In contrast, the parallel upregulation of TLR4 and TLR2 on the surface of monocytes determined by flow cytometry was attenuated by more than half after LPS challenge (P<0.02). Suppressed TLR4 and TLR2 expression was associated with diminished circulating concentrations of tumor necrosis factor-alpha and monocyte chemoattractant protein-1. In conclusion, high-dose simvastatin pretreatment blunted TLR4 and TLR2 expression on monocytes in a human endotoxemia model on a posttranscriptional level. This suppressive effect of statins on key receptors of the innate immunity which was associated with a reduction of effector cytokines reveals a potential mechanism for their beneficial effects in sepsis and cardiovascular disease.

An effective measure to assess zinc status of humans has remained elusive, in contrast to iron, where a number of indicators of metabolism/function are available. Using monocytes, T lymphocytes, and granulocytes isolated by magnetic sorting and dried blood spots (DBS) derived from 50 mul of peripheral blood, we evaluated the response of metallothionein (MT), zinc transporter, and cytokine genes to a modest (15 mg of Zn per day) dietary zinc supplement in human subjects. Transcript abundance was measured by quantitative real-time RT-PCR (QRT-PCR). Zinc supplementation increased MT mRNA abundance by up to 2-fold in RNA from leukocyte subsets, and 4-fold in RNA from DBS. Transcript levels for the zinc transporter genes ZnT1 and Zip3 were increased and decreased, respectively, by zinc supplementation. Expression of the ZnT and Zip genes among leukocyte subsets differ by up to 270-fold. Monocytes and granulocytes from supplemented subjects were activated by LPS, whereas T lymphocytes were activated by mimicking antigen presentation. With zinc consumption, TNF-alpha and IL-1beta expression was greater in activated monocytes and granulocytes, and IFN-gamma mRNA levels were higher in activated T lymphocytes. These studies show that QRT-PCR is a tool to reliably measure transcript abundance for nutritionally responsive genes in human subjects, and that a small sample of whole dried blood, when appropriately collected, can be used as the source of total RNA for QRT-PCR analysis. The results obtained also show that zinc supplementation of human subjects programs specific leukocytic subsets to show enhanced cytokine expression upon activation by stimulators of immunity.

Inflammation contributes to cardiovascular disease and is exacerbated with increased adiposity, particularly omental adiposity; however, the role of epicardial fat is poorly understood.

Summary Background Granulocyte/macrophage colony-stimulating factor (GM-CSF), a cytokine with pleiotropic functions, has been successfully employed in the treatment of chronic skin ulcers. The biological effects underlying GM-CSF action in impaired wound healing have been only partly clarified. Objectives To investigate the effects of GM-CSF treatment of chronic venous ulcers on lesion vascularization and on the local synthesis of the angiogenic factors vascular endothelial growth factor (VEGF) and placenta growth factor (PlGF). Methods Patients with nonhealing venous leg ulcers were treated with intradermal injection of recombinant human GM-CSF, and biopsies were taken at the ulcer margin before and 5 days after administration. Wound vascularization was analysed by immunohistochemistry using antiplatelet endothelial cell adhesion molecule-1/CD31 and anti-alpha-smooth muscle actin antibodies. VEGF and PlGF transcription was assessed by in situ hybridization. To identify the cell populations transcribing VEGF within the ulcer bed, the VEGF hybridization signal was correlated with the immunostaining for different cell type markers on serial sections. Direct induction of VEGF transcription by GM-CSF was investigated in GM-CSF-treated cultured macrophages and keratinocytes. Results Blood vessel density was significantly increased in the ulcer bed following GM-CSF treatment. VEGF transcripts were localized in keratinocytes at the ulcer margin both before and after GM-CSF treatment, whereas a VEGF hybridization signal was evident within the ulcer bed only following administration. PlGF mRNA was barely detectable in keratinocytes at the ulcer margin and was not visibly increased after treatment. Unlike VEGF, a specific PlGF hybridization signal could not be detected in cells within the ulcer following GM-CSF administration. Monocytes/macrophages were the main cell population transcribing VEGF after GM-CSF treatment. In vitro analysis demonstrated that VEGF transcription can be directly stimulated by GM-CSF in a differentiated monocytic cell line, but not in keratinocytes. Conclusions Our data show that increased vascularization is associated with GM-CSF treatment of chronic venous ulcers and indicate that inflammatory cell-derived VEGF may act as an angiogenic mediator of the healing effect of GM-CSF in chronic ulcers.

Polyneuropathy, organomegaly, endocrinopathy, M-protein, and skin changes syndrome is a rare multisystem disorder. Overproduction of vascular endothelial growth factor (VEGF) by plasmocytoma could be responsible for the symptoms. The authors treated four patients with high-dose chemotherapy and autologous peripheral blood stem cell transplantation. Within 6 months, symptoms associated with rapid normalization of serum VEGF levels improved.

Increased levels of reactive oxygen and nitrogen species, as seen in response to exercise, challenge the cellular integrity. Important protective adaptive changes include induction of heat shock proteins (HSPs). We hypothesized that supplementation with antioxidant vitamins C (ascorbic acid) and E (tocopherol) would attenuate the exercise-induced increase of HSP72 in the skeletal muscle and in the circulation. Using randomization, we allocated 21 young men into three groups receiving one of the following oral supplementations: RRR-alpha-tocopherol 400 IU/day + ascorbic acid (AA) 500 mg/day (CEalpha), RRR-alpha-tocopherol 290 IU/day + RRR-gamma-tocopherol 130 IU/day + AA 500 mg/day (CEalphagamma), or placebo (Control). After 28 days of supplementation, the subjects performed 3 h of knee extensor exercise at 50% of the maximal power output. HSP72 mRNA and protein content was determined in muscle biopsies obtained from vastus lateralis at rest (0 h), postexercise (3 h), and after a 3-h recovery (6 h). In addition, blood was sampled for measurements of HSP72, alpha-tocopherol, gamma-tocopherol, AA, and 8-iso-prostaglandin-F2alpha (8-PGF2alpha). Postsupplementation, the groups differed with respect to plasma vitamin levels. The marker of lipid peroxidation, 8-iso-PGF2alpha, increased from 0 h to 3 h in all groups, however, markedly less (P < 0.05) in CEalpha. In Control, skeletal muscle HSP72 mRNA content increased 2.5-fold (P < 0.05) and serum HSP72 protein increased 4-fold (P < 0.05) in response to exercise, whereas a significant increase of skeletal muscle HSP72 protein content was not observed (P = 0.07). In CEalpha, skeletal muscle HSP72 mRNA, HSP72 protein, and serum HSP72 were not different from Control in response to exercise. In contrast, the effect of exercise on skeletal muscle HSP72 mRNA and protein, as well as circulating HSP72, was completely blunted in CEalphagamma. The results indicate that gamma-tocopherol comprises a potent inhibitor of the exercise-induced increase of HSP72 in skeletal muscle as well as in the circulation.