Accordingly, we studied whether the combination of IFN-α and D-fraction (PDF), a bioactive mushroom extract, might potentiate anticancer activity of IFN-α in prostate cancer PC-3 cells in vitro.Prostate cancer remains the most common malignancy among elderly men and the second leading cause of cancer death in the United States. Although several conventional therapies are currently available, they have a low efficacy and the more effective treatment modalities need to be established. Interferons (IFNs) are one of such options known as immunotherapy and demonstrated their antitumor effects on certain cancer types. Yet such antitumor activity should be improved or potentiated to have the satisfactory outcomes. In fact, 2b , PDF , or their combinations were assessed on the growth of PC-3 cells at 72 h. Cell cycle analysis using a flow cytometer and Western blot analysis were performed to explore antiproliferative mechanism of these agents. The dose-dependent study showed that IFN-α2b up to 20,000 (20 K) IU/ml had no significant effects, but >60% growth reduction was attained ≤50 K IU/ml. Similarly, PDF showed no effects up to 250 μg/ml but ~65% growth reduction was seen at 1,000 μg/ml. When IFN-α2b and PDF were combined, a relatively low concentration (10 K IU/ml) of IFN-α2b and PDF (250 μg/ml) resulted in a ~65% growth reduction. This was accompanied by a G1 cell cycle arrest, indicated by cell cycle analysis. Western blots also revealed that the G1-specific cell cycle regulators, CDK2, CDK4, CDK6, cyclin D1, and cyclin E, had been significantly (>60%) down-regulated in IFN/PDF-treated cells.Potential effects of recombinant IFN-α2b (10 K IU/ml) and PDF (250 μg/ml) is capable of inducing a ~65% reduction in PC-3 cell growth. This appears to be due to a synergistic potentiation of two agents, leading to a G1 cell cycle arrest. Thus, it is conceivable that PDF may potentiate IFN-α2b activity, improving immunotherapy for prostate cancer.The combination of IFN-α Current therapy for prostate cancer (CaP), the most common malignancy in elderly men in the United States , is direin vitro and in vivo cancer models [Interferons (IFNs) are known to trigger multiple cellular responses including antiviral activity, growth inhibition, cell differentiation and immunoregulation . Many str models . For insr models -8. Compar models , althougr models . These dGrifola frondosa), is the acid-insoluble, alkali-soluble and hot water-extractable fraction [6 dalton [The D-fraction (PDF), the unique proteoglucan extracted from maitake mushroom  or PDF  for 72 h. Such a dose-dependent study showed that IFN-α2b had no apparent effects up to 20 K IU/ml but induced >60% growth reduction at 50 K and 100 K IU/ml  to be effective.To examine the possible effects of individual IFN-α/ml Fig. . Similar/ml Fig. . Thus, t2b and PDF may exhibit the enhanced growth inhibitory effects, the varying concentrations of IFN-α2b and PDF were combined and their effects on PC-3 cell growth were assessed. Such results showed that combinations of 10 K IU/ml of IFN-α2b and 100 or 250 μg/ml of PDF resulted in nearly 40% or 65% growth reduction, respectively  and PDF (100 or 250 μg/ml) by itself had no such effects , PDF (250 μg/ml), or their combination for 72 h, and they were subjected to cell cycle analysis using a flow cytometer. IFN-α2b or PDF alone had little effects similar to cell cycle phase distribution in control cells; however, the IFN-α2b/PDF combination caused an ~63% decrease in cell number in the S phase with a concomitant 55% increase in the G1-phase cell population compared to those in controls /PDF (250 μg/ml) combination, we also examined its effects on the specific cell cycle regulators for the G1-S phase transition such as CDK2, CDK4, CDK6, cyclin D1, and cyclin E [2b/PDF treatment were all significantly reduced by >60% (quantitated by densitometric scanning), compared to those in controls  somewhat limit its clinical utility. Accordingly, reducing a cost while improving the efficacy of IFNs, "combination" therapy has been proposed and promoted.IFNs belong to the family of cytokines and are capable of activating a cascade of intracellular pathways that regulate cell growth/differentiation and also produce antiviral and immunological responses ,15. Part2b and D-fraction (PDF). Dose-dependent studies showed that IFN-α2b ≥ 50 K IU/ml or PDF at 1,000 μg/ml was capable of inducing >60% growth reduction in prostate cancer PC-3 cells  induced by 50 K IU/ml of IFN-α2b alone, merely "1/5th" (10 K IU/ml) of that IFN-α2b would be needed when combined with PDF. It is then plausible that PDF may not only help potentiate IFN-α2b activity but also help cut the cost down.In the present study, we explored such combination therapy as an alternative approach for prostate cancer immunotherapy; i.e. combination of IFN-α2b/PDF combination on the cell cycle regulation in order to explore the growth inhibitory mechanism. Cell cycle analysis revealed a ~63% decrease in the S-phase cell number with a concomitant 55% increase in the G1-phase cell number following the treatment of IFN-α2b/PDF combination  down-regulated , triggering the specific cascade events  on the cell cycle to ultimately cease cancer cell growth. Therefore, such biochemical studies are undoubtedly required and being underway in our laboratory.Yet, it should be also noted that IFNs are known to modulate many proteins and enzymes , particuight DNA . This maight DNA . DNA-PK ight DNA . These i2b/PDF combination observed in this in vitro study might be also demonstrated in animal study (in vivo). Such study would then allow us to assess the actual efficacy of IFN-α2b/PDF combination on prostate tumor grown in mice and to determine the effective or tolerable physiological concentrations of these agents. This will be conducted shortly as our Phase II study.In addition, it is important to further investigate whether the enhanced growth inhibitory effect of IFN-α2b or PDF in human use would be certainly concerned. IFN-α2b has been often used in immunotherapy for various cancer patients and its concentrations up to 5 × 106 IU have been shown to be relatively safe and tolerable in those with prostate cancer [2b (in combination with PDF) in this study would be extrapolated to actual patients. For PDF, early animal and clinical studies ascertained the safety of PDF without any side/adverse effects [2b and PDF and also obtaining valid information on their effective and tolerable physiological concentrations. It may then help lead us to an ultimate clinical trial in the future.Furthermore, the safety of IFN-αe cancer ,22. It e effects . This wa effects . Althoug2b and PDF demonstrates a synergistic antiproliferative activity on prostate cancer PC-3 cells. This potentiated growth inhibition results primarily from a G1 cell cycle arrest. Therefore, the low-dose IFN-α2b/PDF combination may provide an alternative, improved immunotherapy for prostate cancer, implying its clinical utility/application. It is promising but further studies are yet required.In summary, the combination of IFN-α5 cells/ml and were cultured with recombinant interferon-α2b , D-fraction  or their combinations. Cell numbers were then assessed at specified times using the trypan blue exclusion method.The human prostate cancer PC-3 cells, derived from a patient with bone metastasis, were obtained from the American Type Culture Collection . Cells were maintained in RPMI-1640 medium containing 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 μg/ml). Routinely, culture medium was changed every 3 to 4 days and the passage of cells was performed weekly. For experiments, cells were seeded in T-75 flasks or 6-well culture plates at the initial cell density of 1 × 106 cells were resuspended in 500 μl of propidium iodide solution  and incubated at room temperature for 1 h. Ten thousand nuclei were analyzed for each sample, and CellFit software was used to quantify cell cycle compartments and estimate cell cycle phase fractions.A FACScan flow cytometer , equipped with a double discrimination module, was employed for cell cycle analysis. Approximately 1 × 102b/PDF-treated cells were resuspended in cell lysis buffer and cell lysates were prepared by freeze-thaw three times in liquid nitrogen. The Western blot procedure essentially followed the protocol described previously [2b/PDF-treated cell lysates was resolved by 10% SDS-PAGE (SDS-polyacrylamide gel electrophoresis) and transferred to a nitrocellulose membrane. The blot was first incubated for 90 min with the primary antibodies against CDK2, CDK4, CDK6, cyclin D1, or cyclin E , followed by incubation with the appropriate secondary antibody conjugates for 30 min. The specific immunoreactive proteins were then detected by chemiluminescence, following a vender's protocol , and quantified using a scan densitometer .Cell pellets from control and IFN-αeviously . Brieflyt test. A value of p < 0.05 is considered to be significant.All data are presented as the mean ± SD (standard deviation), and statistical differences between groups were assessed with the unpaired Student's The authors declare that they have no competing interests.PP is a primary investigator in charge of performing all experiments and drafting the manuscript; BL and SR serve as assistants for PP to help set up and run experiments ; MC is the department chairman, providing us with all his support for this project; and SK is responsible for designing experiments, analyzing the data , and editing/finalizing the manuscript. All authors read and approved the final manuscript.


C. elegans, disruption of eitherconjugation or oxidation of 4-HNE leads to fat accumulation, whereasaugmentation of 4-HNE conjugation results in a lean phenotype. Moreover,direct treatment of C. elegans with synthetic 4-HNE causes increasedlipid storage, directly demonstrating a causative role of 4-HNE. Thepostulated mechanism, which involves modulation of acetyl-CoA carboxylaseactivity, could contribute to the triggering and maintenance of the obesephenotype on a purely metabolic level.Deposition and mobilization of fat in an organism aretightly controlled by multiple levels of endocrine and neuroendocrineregulation. Because these hormonal mechanisms ultimately act by affectingbiochemical reactions of fat synthesis or utilization, obesity could bealso modulated by altering directly the underlying lipid biochemistry. Wehave previously shown that genetically modified mice with an elevated levelof the lipid peroxidation product 4-HNE become obese. We now demonstratethat the process is phylogenetically conserved and thus likely to beuniversal. In the nematode  To be able to undergo all three reactions, a substrate must containthe requisite target functional groups; this requirement restricts the set ofpossible substrates to α,β-unsaturated carbonyl compoundssuch as 4‑HNE. Moreover,experimental interventions that lower the expression of 4‑HNE-conjugating GSTs in C. elegans (gst-10)and in mice (mGSTA4-4) resulted in a similar phenotype, i.e., increasedfat storage. The two enzymes are not orthologs, and their ability to conjugate 4‑HNE probably evolved independently. E. coli OP50 weregrown in LB medium .C. elegans overexpressing gst‑10 was described previously  supplemented with cholesterol (3 μl of a 5 mg/ml stock solution in ethanol) and with the same E. coli strain that was used for the initial growth on plates at a final density of A600= 1.0. The cultures were maintained at 20°C except for gst-10 overexpressorswhich were grown at 25°C. The medium was replaced every 12 hours with freshmedium containing the components listed above, including fresh bacteria.For liquid culture, synchronized C. elegans with 4‑HNE.Treatment of  Worms weremaintained in liquid culture for 3 days under the conditions described above,except that 4‑HNE (at concentrations ranging from zero to 2.0 mM) wasadded to the growth medium. Therefore, worms were exposed to fresh 4‑HNEevery 12 hours for a total of three days.C. elegans.Nile red and Sudan black staining of  For Nile red staining, worms weregrown in liquid culture in medium supplemented with 75 ng/ml Nile Red (added asa 0.5 mg/ml stock solution in acetone). After 3 days of treatment with Nile redand, where used, with concurrently added 4‑HNE, worms were washed for 3× 10 min with S-buffer, and were placed on a slide and covered with acoverslip. Randomly selected fields were photographed under a fluorescencemicroscope  using a4× objective and an identical exposure time (300 ms) for all images. Thefilter cube consisted of a 465-495 nm excitation filter, a 505 nm long passdichroic mirror, and a 540 nm long pass emission filter. Fluorescence spectraof Nile red-stained lipids of different polarity  andrandomly chosen fields were photographed under a brightfield microscope  with Nomarski optics and a4× objective. The exposure time (10 ms) was identical for all images.For Sudan black B staining , worms whttp://rsbweb.nih.gov/ij. Files wereconverted to 8-bit depth, were inverted for Sudan black stains, and theperimeter of 50 to 100 non-overlapping, complete worm images was tracedmanually. The mean pixel intensity was recorded for each scored animal afterbackground subtraction. The mean intensity represents the amount of dye, andthus amount of lipid, per unit of surface area in the image, i.e., thedensity of lipid in the organism. This lipid density parameter corresponds tothat used by Ashrafi et al. [All images were saved as monochrome 16-bit TIFF files,and were analyzed in the ImageJ program i et al. , except Enzyme assays. Conjugation of 4‑HNEwith gluta-thione (GST activity) was measured according to Alin et al. [C. elegans obtained as describedpreviously [+-dependent oxidationof 4‑HNE , worms from four 100-mm plates were washed with 50 mM Tris-HCl, pH 7.4, 5 mMEDTA, and stored at -70°C. Immediately before use, 0.1 ml of the above bufferwere added to the worm pellet, and the suspension was sonicated on ice for 3× 10 s and centrifuged for 20 min at 12,000 g at 4°C. Aldehydedehydrogenase activity was assayed in the supernatant according to [+ was used as the co-substrate instead of NADP+.n et al.  inwholeeviously . For therding to , exceptDetermination of metabolite levels. For thedetermination of citrate, frozen worms were homogenized in 1 M perchloricacid. Following centrifugation, the supernatant was neutralized and citratewas measured by a coupled enzyme assay utilizing citrate lyase and malate andlactate dehydrogenases [ogenases , as impl.[To assay malonyl-CoA, worm pellets were frozen andstored at -70°C. On the day of assay, the pellets were pulverized in a mortarat liquid nitrogen temperature, and were lyophilized overnight. The weight ofthe powder was recorded, and the samples were processed and malonyl-CoA wasdetermined by HPLC by a modification of the method of Lazzarino et al. as descr..Assay of 4‑HNE-proteinadducts.The level of 4‑HNE-modifiedproteins was determined by competitive ELISA [ve ELISA  as modifve ELISA .Materials.4‑HNE dimethylacetal wassynthesized according to [rding to ,95. 4‑HN


The current debate about the side effects of induction agents, e.g. possible adrenal suppression through etomidate, emphasizes the relevance of choosing the correct induction agent in septic patients. However, cardiovascular depression is still the most prominent adverse effect of these agents, and might be especially hazardous in septic patients presenting with a biventricular cardiac dysfunction - or so-called septic cardiomyopathy. Therefore, we tested the dose-response direct cardiac effects of clinically available induction agents in an isolated septic rat heart model.-8 to 1 × 10-4 M. Left ventricular pressure, contractility and lusitropy, and coronary flow were measured. Cardiac work, myocardial oxygen delivery, oxygen consumption, and percentage of oxygen extraction were calculated.A polymicrobial sepsis was induced via cecal ligation and single puncture. Hearts (n = 50) were isolated and randomly assigned to five groups, each receiving etomidate, s(+)-ketamine, midazolam, propofol, or methohexitone at concentrations of 1 × 10All of the induction agents tested showed a dose-dependent depression of cardiac work. Maximal cardiac work dysfunction occurred in the rank order of s(+)-ketamine (-6%) <etomidate (-17%) <methohexitone (-31%) <midazolam (-38%) <propofol (-50%). In addition, propofol showed a maximum decrease in contractility of -38%, a reduction in lusitropy of -44%, and a direct vasodilator effect by increasing coronary flow by +29%.Overall, this study demonstrates that these tested drugs indeed have differential direct cardiac effects in the isolated septic heart. Propofol showed the most pronounced adverse direct cardiac effects. In contrast, S(+)ketamine showed cardiovascular stability over a wide range of concentrations, and might therefore be a beneficial alternative to etomidate. Although the ideal induction agent for critically ill patients has not yet been found, there is general agreement that in those patients an induction agent that provides cardiovascular stability upon induction of anesthesia would be first choice. Nevertheless, current guidelines do not recommend one induction agent over another ,2. HowevHowever, the most significant adverse effect of induction agents is cardiovascular depression, which has already been well described in healthy animal models and humans after intravenous administration. The degree of negative cardiovascular effects depends on dose and speed of administration and appears to vary greatly among the commonly used drugs ,6. In noThe cardiovascular dysfunction in sepsis derives from a reduced systemic vascular resistance typically complicated by decreased cardiac function ,2. This in vitro effects of these agents on cardiac contractile function in sepsis, and the isolated, dose-dependent effects of these induction agents on myocardial excitability, contractility, coronary flow, and oxygen utilization in a septic heart are still unknown. We used the isolated ex vivo heart model to study the direct cardiac effects in the absence of confounding neurohormonal, metabolic, or systemic factors.However, global cardiac mechanical and metabolic effects of these induction agents in septic cardiomyopathy have thus far not been systematically compared in a dose-dependent fashion. There is very little evidence on the direct Therefore, the aim of this study was to directly compare electrical, mechanical, and metabolic effects of etomidate, s(+)-ketamine, midazolam, propofol, and methohexitone at equimolar concentrations, with special emphasis on their impact on cardiac work.®, Munich, Germany) and had the following composition: Na+ 140 mM; K+ 4.5 mM; Mg2+ 1.2 mM; Ca2+ 2.5 mM; Cl- 134 mM; HCO3- 15.5 mM; H2PO4- 1.2 mM; EDTA 0.05 mM; glucose 11.5 mM; pyruvate 2 mM; mannitol 10 mM; and insulin 5 U/L. Mean aortic inflow pH, partial pressure of carbon dioxide (pCO2), and partial pressure of oxygen (PO2) were 7.39 ± 0.01, 36 ± 1 mmHg, and 580 ± 25 mmHg, respectively. Perfusate and heart temperature was maintained at 36.9 ± 0.3°C throughout the experiment.Approval from the Institutional Animal Care Committee of the University of Goettingen was obtained before initiation of this study. All experimental procedures conformed with German animal safety regulations. Fifty male Wistar rats (weighing 245 ± 3 g) were injected intraperitoneally with 100 mg/kg ketamine and 2.5 to 5 mg/kg xylazine hydrochloride. A polymicrobial sepsis was induced via cecal ligation and a single puncture as reported previously in detail . After 2®, Roche Diagnostic, Mannheim, Germany). Oxygen delivery, percent oxygen extraction, and myocardial oxygen consumption were calculated as noted previously [Spontaneous atrial rate, atrio-ventricular conduction time, and systolic left ventricular pressure (LVP) and its derivative were measured as detailed previously . Coronareviously ,13. Cardeviously . All mea®, AstraZeneca, Wedel, Germany), midazolam , s(+)-ketamine (S(+)-Ketamine®, Pfizer Pharma, Berlin, Germany), methohexitone , or etomidate . Each heart was perfused, in randomized order, at concentrations of 10-8 to 10-4 M with one of these drugs for a period of 15 minutes. There was a 20-minute drug-free washout period. Prior to this study we tested higher concentrations for each of these drugs. However, at concentrations higher than 10-3 M some hearts showed cardiac arrest. Therefore, concentrations of 10-3 M or more were not included in the present study.The experimental protocol is shown in Figure -8 to 10-4 M), which are equivalent to 0.002 to 18 μg/mL propofol (molecular weight: 178.3 mM), 0.003 to 33 μg/mL midazolam (325.8 mM), 0.003 to 27 μg/mL s(+)-ketamine (274.2 mM), 0.003 to 26 μg/mL methohexitone (262.3 mM), and 0.002 to 24 μg/mL etomidate (244.3 mM), correspond to approximate therapeutic plasma-free values  of 5.1 to 11 × 10-7 (97 to 98%) propofol, 3.7 to 37 × 10-9 M (94 to 95%) midazolam, 3.2 to 19 × 10-6 M (12 to 30%) s(+)-ketamine, 4.6 to 9.1 × 10-6 M (70 to 73%) methohexitone, and 0.9 to 4.7 × 10-7 M (77 to 94%) etomidate [The concentrations tested in our study  against the initial control (CTRL). 2)/myocardial oxygen consumption (MVO2) ratio 1.5 ± 0.0 were statistically different from control values of septic hearts. Sham-operated hearts showed control values of etomidate, s(+)-ketamine, midazolam, propofol, and methohexitone in septic hearts were not statistically different between the groups. After a washout period, each parameter returned to baseline level.Control values of sham-operated hearts  3275 ± 84 mmHg/sec, LVP relaxation (-dLVP/dt) 2629 ± 74 mmHg/sec, cardiac work 36036 ± 639 mmHg/beats, and myocardial oxygen supply  and at 1 × 10-5 to 1 × 10-4 M for midazolam (maximum decrease: -47 ± 5%). Reduction of heart rate by midazolam at 1 × 10-4 M was significantly more pronounced compared with all other tested induction agents. Maximum decreases in heart rate were -12 ± 3% for s(+)-ketamine (1 × 10-4 M) and -11 ± 4% for methohexitone (1 × 10-4 M). Only etomidate showed no chronotropic effect at any tested concentration in this study.The comparative effects of etomidate, s(+)-ketamine, midazolam, propofol, and methohexitone on heart rate are shown in Figure -4 M and -19 ± 5% at 1 × 10-4 M was significant for propofol and methohexitone, respectively. The effects of propofol were significantly more pronounced compared with all other agents tested at equimolar concentrations. Other induction agents showed a maximum decrease in contractility of -5 ± 6% for etomidate at 1 × 10-5 M, and a maximum increase in contractility of +7 ± 5% for s(+)-ketamine at 1 × 10-4 M, and +9 ± 6% for midazolam at 1 × 10-6 M. As shown in Figure 4, etomidate, midazolam, methohexitone, and propofol showed negative lusitropic effects with maximal decreases in -dLVP/dt of -7 ± 6% , -21 ± 5% , -21 ± 6% , and -44 ± 5% , respectively. At 1 × 10-4 M the negative reduction of lusitropy by propofol was significantly different compared with all other tested induction agents. In contrast, at 1 × 10-4 M s(+)-ketamine demonstrated an increase in lusitropy of +14 ± 6%. There was a significant difference compared with propofol and midazolam at equimolar concentration.All tested induction agents showed a dose-dependent decrease in cardiac contractility except for midazolam and s(+)-ketamine Figure . The max-4 M by etomidate (maximum decrease: -17 ± 6%), s(+)-ketamine (-6 ± 6%), midazolam (-38 ± 7%), propofol (-50 ± 6%), and methohexitone (-31 ± 4%) in a dose-dependent fashion. At this concentration, the reduction of cardiac performance was significantly different for propofol, midazolam and methohexitone compared with s(+)-ketamine. Additionally, propofol significantly decreased cardiac work at 1 × 10-5 M by -17 ± 4%.Cardiac work Figure  - the pr2/MVO2 ratio -ketamine, midazolam, and methohexitone showed no direct effects on coronary flow, myocardial oxygen supply and demand . Therefore, DOo Figure  was not o Figure  of +58 ±The study was designed to compare the direct effects of five commonly used intravenous induction agents by analyzing cardiac responses at equimolar concentrations in septic hearts. The tested drugs demonstrate differential direct effects on electrical properties, myocardial function, andoxygen supply-to-demand ratio. Propofol showed the most pronounced adverse direct cardiac effects, whereas s(+)ketamine was most beneficial, as it showed cardiac functionality over a wide range of concentration.-8 to 10-5 M in septic hearts. However, at higher concentrations it markedly depresses cardiac work. These concentrations can easily be achieved either by bolus administration or by long-term infusion in patients with severe sepsis or septic shock, especially with multiple-organ failure accompanied by a decreased hepatic and renal metabolism [There are concerns regarding the application of etomidate in critically ill patients, especially in septic patients due to possible adrenal suppression . The inctabolism . Therefotabolism .2+ current (ICa2+), which play an important role in the force of contraction and spontaneous firing of sinoatrial node cells, as demonstrated in electrophysiological experiments [In contrast, s(+)ketamine showed cardiac functionality over a wide range of concentrations. S(+)ketamine is an optical isomer of ketamine and exhibits stereoselective bindings to different receptors, accounting for its three to four times higher anesthetic potency compared with the R(-)-isomer ,22. The eriments .2+ flux [2+ handling or altered calcium transient properties described in septic hearts might be attributable to these differing results [-4 M. This effect suggests that coronary autoregulation was inhibited at this concentration, and propofol may cause a substantial coronary vasodilation when used as an anesthetic induction agent [Midazolam and methohexitone, together with propofol, showed the most adverse effects on cardiac stability. Propofol, midazolam and methohexitone decreased cardiac work in a dose-dependent fashion. At very similar concentrations, Stowe and colleagues showed a decrease in contractility in guinea pig hearts from midazolam, propofol, and thiopental . However2+ flux , the dys results ,25. The on agent ,13. In cex vivo approach and isolated hearts and focused on the direct cardiac effects of the applied induction agents. The advantages of this method are to measure mechanical and metabolic properties in the absence of the confounding effects of other organs, systemic circulation, and a host of peripheral complications such as circulating neurohormonal factors [-5 M, the possible influence of a force-frequency relationship has to be kept in mind when interpretating the presented results.We recognise the limitations of this study. Although the applied sepsis method has the advantages of inducing a 'natural' course of infection, it has limitations with regard to noteworthy outcome variability ,27. In c factors . One potIn conclusion, this study showed that the tested drugs - etomidate, s(+)-ketamine, midazolam, propofol, and methohexitone - indeed have differential direct cardiac effects, even in the isolated septic heart. Propofol showed the most pronounced adverse direct cardiac effects, while S(+)ketamine demonstrated cardiac stability over a wide range of concentrations. Thus, if our data can be extrapolated to apply to humans, it seems that there are alternatives to etomidate such as s(+)ketamine, which demonstrates similar cardiac stability, but with less non-cardiovascular side effects affecting the outcome of septic patients.• Induction agents show differential direct cardiac effects in septic cardiomyopathy.• propofol show most pronounced adverse effects.• S(+)ketamine demonstrates cardiac stability over a wide range of concentrations.2: myocardial oxygen supply; LVP: Left ventricular pressure; MVO2: myocardial oxygen consumption; pCO2: partial pressure of carbon dioxide; PO2: and partial pressure of oxygen.+dLVP/dt: left ventricular contractility; -dLVP/dt: left ventricular relaxation; DOOn behalf of my co-authors I attest that the work has not been funded by any source(s) other than described in the statement. No author or participant has any financial interest in the subject matter, materials or equipment discussed or in competing materials. The laboratory in which the research was performed has not been funded by, or has any participant in the planning, conduct, or reporting of the research been funded by or have financial interests in any source with a real or potential interest in the subject matter, materials, equipment or devices discussed or in any competing product or subject. And the laboratory in which the work was performed or any of the authors or participants have not been funded by any foundation or other non-governmental source that has received funding from any organization with a real or potential interest in the subject matter, materials, equipment or devices discussed, or in any competing product or subject.YZ and BG originated the idea and performed preliminary experiments. HB continued to perform the experiments. BS coordinated to the laboratory support. YZ and WZ were responsible for writing the paper. DL, BS and BG supported the editing of the manuscript and added important comments to the paper. All authors read and approved the final manuscript.The data was presented in part at the 3rd International Congress of the German Sepsis Society in Weimar from 5 to 8 September, 2007.


A total of 37 amplicons were used to screen single nucleotide polymorphisms (SNPs) on 6 F1 bulls. Among 36 SNPs detected in 11 of these 13 genes, 19 were selected for genotyping by the Sequenom assay design on all F2 progeny. Single-marker analysis revealed seven SNPs in ATP binding cassette A1, apolipoproteins A1, B and E, phospholipid transfer protein and paraoxinase 1 genes significantly associated with nine phenotypes (P<0.05). Previously, we reported genetic networks associated with 19 complex phenotypes based on a total of 138 genetic polymorphisms derived from 71 known functional genes. Therefore, after Bonferroni correction, these significant (adjusted P<0.05) and suggestive (adjusted P<0.10) associations were then used to identify genetic networks related to the RCT pathway. Multiple-marker analysis suggested possible genetic networks involving the RCT pathway for kidney-pelvic-heart fat percentage, rib-eye area, and subcutaneous fat depth phenotypes with markers derived from paraoxinase 1, apolipoproteins A1 and E, respectively. The present study confirmed that genes involved in cholesterol homeostasis are useful targets for investigating obesity in humans as well as for improving meat quality phenotypes in a livestock production.In the present study, thirteen genes involved in the reverse cholesterol transport (RCT) pathway were investigated for their associations with three fat depositions, eight fatty acid compositions and two growth-related phenotypes in a Wagyu x Limousin reference population, including 6 F PON1) gene product Reverse cholesterol transport (RCT) pathway represents an important process involved in cholesterol homeostasis In circulation, the activities of lipoprotein lipase (LPL), endothelial lipase (LIPG), and hepatic lipase (LIPC) continuously remodel lipoproteins, which profoundly affect their metabolic fate. The primary function of LPL is to hydrolyze triglyceride-rich lipoproteins, especially chylomicrons and VLDL, thereby generating free fatty acids and glycerol for energy metabolism and storage All these molecular events indicate that the RCT pathway is a major component of lipid homeostasis affecting lipid phenotypes. Mammals achieve intravascular lipid transport though production and metabolism of lipoproteins, which distribute cholesterol and fatty acids to peripheral tissues expressing the appropriate lipases/receptors. However, little effort has been directed towards understanding how the RCT pathway affects different fat depot storages, fatty acid compositions and overall body growth. Furthermore, differences in dietary intake and digestive physiology between ruminants and non-ruminants produce questions regarding the importance of these pathways to cattle. To this end, the aim of the present study was to investigate the potential molecular links of the RCT pathway genes with the fat deposition, fatty acid composition and body growth-related phenotypes using cattle as a model organism.1 bulls, 113 F1 dams, and 246 F2 progeny in the Fort Keogh Livestock and Range Research Laboratory, ARS, USDA. Fat deposition for three depots was measured by a trained evaluator after 48 h of chilling at 2°C and included beef marbling score (BMS), subcutaneous fat depth (SFD) and percent kidney-pelvic-heart fat (KPH). BMS reflects the dispersion of intramuscular fat in the longissimus muscle and is determined subjectively based on U.S. Department of Agriculture standards (http://www.ams.usda.gov). SFD was measured at the 12th to 13th rib interface perpendicular to the outside surface at a point three-fourths the length of the longissimus muscle from its chine bone end. KPH was subjectively estimated as percentage of the carcass weight. In addition, carcass weight (CW) and rib-eye area (REA) were collected as two growth-related phenotypes on these F2 animals. CW was determined as the unchilled weight in pounds immediately after harvest before rinsing/washing and chilling. The area of the longissimus muscle measured in square inches at the 12th rib interface on the beef forequarter was recorded as REA.A Wagyu x Limousin reference population was jointly developed by Washington State University and the Fort Keogh Livestock and Range Research Laboratory, ARS, USDA, as previously described longissimus dorsi muscle tissue were completely saponified with 4.0 ml of 1.18 M KOH in ethanol at 90°C. After about 45 minutes, 2.0 ml of water were added. Cholesterol (CHOL) was extracted with 2.0 ml of hexane, which contained 0.1 mg/ml of stigmasterol as an internal standard for the cholesterol assay. One millilitre of concentrated HCl was added to the original tubes and fatty acids were extracted in 2.0 ml of hexane for fatty acid methyl ester (FAME) preparation using methanolic HCl as a catalyst. The amount of conjugated linoleic acid (CLA) was measured using acid catalysts. FAME data were used to measure the following: saturated fatty acids (SFA)  =  myristic + pentadecanoic + palmitic + heptadecanoic + stearic, monounsaturated fatty acids (MUFA)  =  myristoleic + pentadecenoic + palmitoleic + heptadecenoic + oleic + vaccenic, and polyunsaturated fatty acids (PUFA)  =  linoleic + linolenic. The relative amount of SFA, MUFA and PUFA was defined as SFA  =   ×100%, MUFA  =   ×100% and PUFA  =   ×100%, respectively. Three stearoyl-CoA desaturase activities were estimated as R1  =  (14∶1/14∶0) ×100%, R2  =  (16∶1/16∶0) ×100% and R3  =  (18∶1/18∶0) ×100%.Fatty acid composition in muscle samples was measured according to methodology previously described ABCA1, APOA1, APOB, APOC2, APOE, LCAT, LDLR, LIPC, LIPG, LPL, PLTP, PON1 and SCARB1 were selected for the present study. As discussed above, these genes are involved in the RCT pathway. Manual annotation of each gene occurred as follows: First, cDNA sequences of candidate genes were retrieved from the National Center for Biotechnology Information (NCBI) Entrez database. To produce full length cDNA sequences, the retrieved sequences were re-annotated using electronic rapid amplification of cDNA ends (e-RACE) 1 bulls was amplified in a final volume of 10 µl that contained 12.5 ng of each primer, 150 µM dNTPs, 1.5 mM MgCl2, 50 mM KCl, 20 mM Tris-HCl and 0.25 U of AmpliTaq Gold polymerase . PCR conditions were as follows: 95°C for 10 minutes, 8 cycles of 94°C for 30 sec, 71°C for 30 sec, and 72°C for 30 sec, followed by 37 cycles of 94°C for 30 sec, 59°C for 30 sec, and 72°C for 30 sec, and completed by an extension step at 72°C for 10 min. PCR amplicons were sequenced on a capillary sequencer by High-Throughput Sequencing Solutions . Selected mutations were genotyped in 246 F2 animals using the Sequenom iPLEX assay service provided by Genomics Center at University of Minnesota.A total of 13 genes, including ijklmy is phenotypic measurement of a quantitative trait for animal m, iyear is the effect of the i-th harvest year , jsex is the effect of the j-th sex category , ksires is the effect of the k-th sire producing animal m , age is a covariate for age in days of the animal at harvest, and β is the coefficient vector corresponding to the covariate age, lgenotype represents the effects of each genotype at the l-th SNP locus, for ijklmε is a residual term pertaining to animal m. When L = 1, the above model reduces to single-marker analysis, and the P value is adjusted using Bonferroni correction k is the number of parameters, and L is the maximized value of the likelihood function for the estimated model. Given a data set, several competing models may be ranked according to their AIC, with the best model having the lowest AIC.The HAPLOVIEW ABCA1, 2 in APOA1, 4 in APOB, 1 in APOC2, 8 in APOE, 1 in LCAT, 3 in LIPC, 1 in LIPG, 2 in PLPT, 4 in PON1 and 2 in SCAB1, respectively. Nineteen of the 36 SNPs underlined in As shown in APOA1 - AAFC03114751.1: c11919T>G. The PLTP - AAFC03071797.1: g13994G>T marker was in Hardy-Weinberg disequilibrium (P<0.05). Therefore, these two markers were excluded from further analysis. As a consequence, the HAPLOVIEW analysis was only performed on four markers in ABCA1, five SNPs in APOE and two markers in SCARB1 (ABCA1 markers: 73024G>A and 73157C>T (2r = 100%)    received no calls for n SCARB1 . Strong  = 100%) , between89%–97%)  and betw2 = 95%) . LinkageABCA1 - AAFC03037127.1:c27113G>A with CLA and AAFC03121742.1:g43352C>G with SFD and SFA, APOA1 - AAFC03114751.1:g11357G>A with CW and REA, APOB - AAFC03076821.1:g39163G>A with CHOL, APOE - AAFC03034452.1:g11400G>A with SFD, PLTP - AAFC03071797.1:c13579C>T with CLA and PON1 - AAFC03037852.1:g64283A>G with KPH, respectively (P<0.05) and three were suggestive (adjusted P<0.10).Single-marker analysis revealed seven SNPs significantly associated with nine phenotypes (P<0.05), including ectively . In otheectively , one forectively , one forectively , one forectively , two forectively , one forectively , and oneectively . After BAPOA1 - AAFC03114751.1:g11357G>A had additive effects on both REA : three with additive , three wboth REA  and CW ; the former gene with dominant effect while the latter gene with additive effect  network (model C2) for SFD as an alternative to a three-gene (TFAM-BAK1-CAPN1) network (model C1) identified previously (APOE1) and one with additive effect (BAK1), while the all three genes in previously reported network had additive effects. The difference of AIC between model C1 and model C0 and between model C2 and model C0 were −15.11 and −14.32, respectively. Thus, the AIC-based model comparison strongly supported models C1 and C2 over the base model C0, but the difference of AIC between models C1 and C2 were not decisive. Overall, our results showed that the RCT pathway is mainly involved in fat deposition (KPH and SFD) and muscle growth (REA).The present multiple-marker analysis incorporated markers derived from the RCT pathway and produced a two-gene (eviously . The newLDLR, leading to increased levels of circulating LDL 2 reference population. Obviously, the pathway-based candidate gene approach conducted in this study provides a fast and direct way to determine the genetic variation that underlies complex phenotypes. At the same time, our study also confirmed that genes involved in cholesterol homeostasis are useful targets for investigating obesity in humans Theoretically, lipoprotein pathways contain excellent candidate genes for obesity-related traits because of their direct involvements in transporting cholesterol, triglycerides, and fatty acids to and from peripheral tissues. Other important lipid components are decidedly interconnected with levels of circulating lipoproteins, including saturated fat, which reduces expression of the ApoE mutations have on intramuscular cholesterol are less apparent. Although skeletal muscle cells attain fatty acids via LPL mediated hydrolysis of circulating lipoproteins APOB - AAFC03076821.1:g39163G>A was associated with muscle cholesterol levels in an overdominant QTM mode. Furthermore, this marker failed to be incorporated in the genetic network for amount of cholesterol in muscle. This finding is an interesting contrast to the high level of significance found among other lipid traits and might be evidence that intracellular cholesterol is reliant on mechanisms independent of blood pathways.Up to date, the vast majority of research to understand the genetics of lipoprotein and lipid homeostasis has been directed toward blood lipids. Human ApoE for example, contains two very well known and common SNPs at amino acid positions 112 and 158, which are associated with decreased HDL, increased LDL, and increased plasma cholesterol in circulation longissimus muscle. Unfortunately, our present study could not provide any evidence to support the hypothesis: none of the sequence variations in genes associated with the RCT pathway was associated with variation in marbling. In contrast, APOA1 - AAFC03114751.1:g11357G>A impacted REA, which is a measurement of the size of the longissimus muscle. This means that APOA1 gene might be involved in regulation of muscle growth. In particular, both APOA1 and FABP3 genes combine to affect REA. Just recently, Teltathum and Mekchay APOA1 and FABP3 genes are involved in muscle development and growth.Noro and Kobayashi In summary, our present study revealed that sequence variations of genes in the RCT pathway are associated with KPH, REA and SFD. Both KPH and SFD are phenotypes related to fat deposition, while REA is connected to muscle growth. From the livestock production point of view, the SNPs evaluated in the present study are strong candidates to join existing panels for marker-assisted selection of meat quality phenotypes in beef cattle. The markers identified in the present study might also have implications beyond the field of animal breeding and improvements and be directive in RCT pathway-related disease research.


Ingestion of carbohydrate (CHO) and protein (PRO) following intense exercise has been reported to increase insulin levels, optimize glycogen resynthesis, enhance PRO synthesis, and lessen the immuno-suppressive effects of intense exercise. Since different forms of CHO have varying glycemic effects, the purpose of this study was to determine whether the type of CHO ingested with PRO following resistance-exercise affects blood glucose availability and insulin levels, markers of anabolism and catabolism, and/or general immune markers.40 resistance-trained subjects performed a standardized resistance training workout and then ingested in a double blind and randomized manner 40 g of whey PRO with 120 g of sucrose (S), honey powder (H), or maltodextrin (M). A non-supplemented control group (C) was also evaluated. Blood samples were collected prior to and following exercise as well as 30, 60, 90, and 120 min after ingestion of the supplements. Data were analyzed by repeated measures ANOVA or ANCOVA using baseline values as a covariate if necessary.uIU/mL), H (150.1 ± 25.39 uIU/mL), and M (154.8 ± 18.9 uIU/mL) were greater than C (8.7 ± 2.9 uIU/mL) as was AUC with no significant differences observed among types of CHO. No significant group × time effects were observed among groups in testosterone, cortisol, the ratio of testosterone to cortisol, muscle and liver enzymes, or general markers of immunity.Glucose concentration 30 min following ingestion showed the H group (7.12 ± 0.2 mmol/L) to be greater than S ; M , and C  groups. No significant differences were observed among groups in glucose area under the curve (AUC) values, although the H group showed a trend versus control (p = 0.06). Insulin response for each treatment was significant by time (p < 0.0001), treatment (p < 0.0001) and AUC (p < 0.0001). 30-min peak post-feeding insulin for S (136.2 ± 15.6 CHO and PRO ingestion following exercise significantly influences glucose and insulin concentrations. Although some trends were observed suggesting that H maintained blood glucose levels to a better degree, no significant differences were observed among types of CHO ingested on insulin levels. These findings suggest that each of these forms of CHO can serve as effective sources of CHO to ingest with PRO in and attempt to promote post-exercise anabolic responses. It has been recommended that athletes ingest CHO and PRO following exercise in order to enhance glycogen resynthesis, promote an anabolic hormonal environment, enhance PRO synthesis, and/or lessen the immuno-suppressive effects of intense exercise -5. TheseWhile a number of studies have evaluated the effects of providing different forms of PRO and amino acids on recovery and/or training adaptations ,16,23-27Forty subjects  volunteered to participate in this study. All subjects had participated in at least one year of resistance training prior to testing and were informed of the possible risks of the investigation before giving their written and informed consent previously approved by the University of Memphis Institutional Review Board for the use of human subjects.Nautilus, Inc., Vancouver, WA, USA) weight-training exercises used during the experimental treatment. Exercises included the chest press, seated row, shoulder press, lat pull, leg extension, leg curl, biceps curl, triceps extension, and leg press. For the exercises in which 1RM was exceeded by the weights available on an individual machine, the Epley formula was used to predict 1RM based on the number of repetitions lifted at a given weight [Prior to the start of this trial, a familiarization session was conducted to obtain one-repetition maximum (1RM) for each of nine Nautilus (n weight . Rest peSubjects reported to the testing laboratory after abstaining from exercise for 48-hours prior to testing and fasting overnight (~12 h). The time of day was standardized within a 2-h starting time for all subjects in order to minimize potential diurnal variation in hormonal concentrations. Upon arrival weight (kg) and height (cm) were recorded. Subjects then donated a pre-exercise baseline blood samples prior to starting the resistance-training workout. Each subject performed 3 sets of 10 repetitions at approximately 70% of 1 RM on nine exercises as described above. Each set of exercise was interspersed with a 2-minute rest period and research assistants monitored all sessions. If a participant could not complete the full 10 repetitions the weight was reduced so 10 repetitions could be completed during the following set of exercise. During each set, the weight lifted and the number of repetitions performed was recorded for each subject in order to calculate total lifting volume. Following the completion of the workout, subjects returned to the laboratory and donated a post exercise blood sample.ADM Arkady, Olathe, KS, USA) contained a 95% mixed CHO source containing fructose (31.5%), glucose (26%), wheat starch (25.3%), soluble fiber (12.5%) and maltose (4.7%). The supplements were similarly colored, flavored and packaged for double-blind administration by an independent food-packaging lab . Research assistant's blended pre-measured volumes of the powder into 16 ounces of water to form a milk shake type drink. Subjects were given as much time as needed to ingest the supplements which typically was less than 5-min. Once the subject consumed the supplement, a timer was started and blood samples were taken at 30, 60, 90, and 120-min during recovery. A similar time frame for collection of blood samples was employed for the control group. Subjects remained seated during the recovery period. As blood samples were collected, subjects were asked to respond to a questionnaire assessing the severity of hypoglycemia, dizziness, fatigue, headache, and stomach upset they experienced throughout the experiment. Questions were asked on a scale of 0–10 with 0 having no symptoms and 10 being most severe.After the post-exercise blood sample was obtained, subjects received in a double blind and randomized manner a CHO and PRO supplement containing 40 g of whey PRO with 120 g of sucrose (S), powdered honey (H), or maltodextrin (M). The remaining group served as a non-supplemented control group. These forms of CHO were selected because prior research in our lab demonstrated that ingesting 50 g of gel forms of these CHO's resulted in significantly different glucose and insulin profiles . The H pJohnson & Johnson Medical, Arlington, TX, USA). Once the catheter was inserted and stabilized, a locking luer male adapter plug with an intermittent injection site was then connected to the female end of the catheter. A Vacutainer™ needle  was then attached to a Vacutainer holder in which the needle was inserted into the injection site of the adapter plug. Approximately 15 mL of venous blood was collected into two separate 6 mL labeled Vacutainer brand SST™ tubes and 5 mL into an EDTA Vacutainer tube . Once the blood samples were obtained, approximately 2–3 mL of Bacteriostatic 0.9% sodium chloride  was infused into the catheter line using a syringe. Once the line was cleared with saline, the catheter was locked to prevent clotting of blood in the line between sampling intervals.During the pre-exercise baseline blood collection period, blood samples were obtained by a qualified nurse/phlebotomist using standardized venipuncture techniques. Following exercise, subjects were fitted with a 20G × 1" Jelco™ intravenous catheter . One of the SST tubes was divided into four labeled 2.0 mL Costar micro centrifuge tubes  and frozen in a So-Low Ultra freezer  at -80°C for subsequent hormonal analysis. The second SST tube and an EDTA tube were sent to Quest Diagnostics  for analysis of glucose, muscle and liver enzymes. Serum samples were assayed using a Technicon DAX model 96-0147 automated chemistry analyzer  following standard clinical procedures. Whole blood cell counts with percent differentials were run on whole blood samples using a Coulter STKS automated analyzer using standard procedures . These analyzers were calibrated daily to controls according to manufacturer's recommendations and federal guidelines for clinical diagnostic laboratories. Test to test reliability of performing these assays ranged from 2 to 6% for individual assays with an average variation of ± 3%. Samples were run in duplicate to verify results if the observed values were outside control values and/or clinical norms according to standard procedures. Blood samples were assayed for each variable at all data points with the exception that creatine kinase was only measured at baseline, following exercise, and 120 minutes following supplementation.Blood samples were then centrifuged for 10 minutes at 3000 rpm in an Adams Physicians Compact Centrifuge . All insulin assays were completed in duplicate and read at 450 and 600 nm with an MRX Microplate Reader  in ambient conditions averaging 27°C and 23% relative humidity. Outlying concentrations with coefficients of variability > 10% were removed to provide the best fitting curve. The intra-assay coefficient of variability for insulin measurements was 3.4% while the inter-assay coefficient of variability was 5.2%. The best fitting curve provided an r2 value of ≥ 0.9916 for insulin. Testosterone and cortisol were assayed in the same manner as insulin using ACTIVE™ DSL-10-4000 and DSL-10-2000 Enzyme Immunoassay (EIA) kits, respectively. The testosterone and cortisol mean coefficient of variability was 2.6% and 2.3% respectively. Inter-assay coefficient of variability for testosterone and cortisol were 13.8% and 11%, respectively. The best fitting curve provided an r2 value of ≥ 0.9996 for testosterone and ≥ 0.9668 for cortisol.Frozen serum samples were assayed at the Exercise Biochemistry Lab at the University of Memphis using standardized spectrophotometric and enzymatic immunoassay procedures. Each sample was thawed only once and decoded only after all the analyses were completed. The quantitative measurement of insulin was determined using the DSL-10-1600 ACTIVE™ insulin Enzyme-Linked Immunosorbent (ELISA) Kit . A three-way multivariate analysis of variance (MANOVA) was utilized to examine gender × group × time interactions. As noted below, although some gender effects were observed, no significant group × time × gender differences were observed. Therefore, two-way repeated measures analysis of variance (ANOVA) was utilized to evaluate treatment, time, and treatment × time interactions. Least square significant difference (LSD) post-hoc analyses were used when significant ANOVA effects were observed. For values that where significant between groups at baseline, repeated measures ANCOVA analyses were employed. Consequent to randomization procedures, total lifting volume, fasting glucose and insulin concentrations were different between groups. Thus, covariate adjustments were made to the repeated measures analysis and AUC measures using these three variables. Delta concentrations were also calculated on post-exercise responses by subtracting 30, 60, 90, and 120-minute concentrations from the immediate un-fed/post-exercise concentration for glucose, insulin, testosterone and cortisol so as to determine integrated area under the curve (AUC) via trapezoidal rules. Data are presented as means and ± SEM.Data were analyzed using Statview for Windows , seated row (1.37 ± 0.06 kg·kg-1), shoulder press (0.70 ± 0.05 kg·kg-1), lat pull (0.79 ± 0.03 kg·kg-1), leg extension (1.0 ± 0.06 kg·kg-1), leg curl (0.51 ± 0.03 kg·kg-1), biceps curl (0.62 ± 0.03 kg·kg-1), triceps extension (0.66 ± 0.04 kg·kg-1) and leg press (2.0 ± 0.1 kg·kg-1). After adjustments were made during the exercise treatment in order to complete 3 sets of 10 repetitions per exercise, the mean percentage of maximum weight lifted during the weight training stimulus were as follows: chest press (66.6% ± 1.5), seated row (69.1% ± 1.1), shoulder press (62.1% ± 1.3), lat pull down (69.5% ± 0.7), leg extension (64.5% ± 2.0), leg curl (60.8% ± 2.3), biceps curl (64.3% ± 1.1), triceps extension (67.7% ± 1.0), and leg press (67.9% ± 1.3). The total lifting volume for each treatment group was: control , sucrose , honey , and maltodextrin . No significant differences were observed among groups in total lifting volume (p = 0.18).Demographic data for each treatment group are presented in Table Gender analysis revealed that testosterone (p = 0.001) and the ratio of testosterone to cortisol (p = 0.001) values were significantly lower in women than men. However, no gender differences were observed in cortisol (p = 0.33). Likewise, no group × gender interactions were observed for cortisol (p = 0.57), testosterone (p = 0.77), or the ratio of testosterone to cortisol (p = 0.43). Moreover, no significant group × time × gender interactions were observed in cortisol (p = 0.10), testosterone (p = 0.88) or the ratio of cortisol to testosterone (p = 0.54). Therefore, data were analyzed by a two-way ANOVA and are presented as means for men and women combined.Figure Between-group treatment effects were also noted for plasma glucose concentrations (p < 0.056) as post-hoc analysis revealed 30 min glucose concentrations for the H group (7.12 ± 0.2 mmol/L) to be greater than S ; M , and C . At 60 min following ingestion, the H group was still greater than the S group (p < 0.02). The M group was also greater than C 30 min following ingestion (p < 0.03). No between group differences were observed at 90 and 120 min. Figure uIU/mL), H (150.1 ± 25.3 uIU/mL), and M (154.8 ± 18.9 uIU/mL) were greater than C (8.7 ± 2.9 uIU/mL) as was the change in AUC after ingestion of the supplements . However, the type of CHO ingested with PRO did not significantly affect insulin response.Significant effects for time (p < 0.0001), treatment (p < 0.00001) and AUC (p < 0.0001) were observed in insulin values. Insulin concentrations (p < 0.05) and insulin AUC values (p < 0.05) were significantly higher than C values at 30, 60 90 120 min (p < 0.05) in the S, H, and M groups. 30-min peak post-feeding insulin for S (136.2 ± 15.6 Table Table Table Table No significant differences were observed among groups in perceptions of hypoglycemia (p = 0.851), dizziness (p = 0.711), headache (p = 0.422), stomach upset (p = 0.325), or fatigue (p = 0.837).Ingestion of CHO and PRO following intense exercise has been reported to increase insulin levels, optimize glycogen resynthesis, enhance PRO synthesis, and lessen the immuno-suppressive effects of intense exercise ,14,16,35uIU/mL; S 136.2 ± 15.6 uIU/mL; H 150.1 ± 25.3 uIU/mL; M 154.8 ± 18.9 uIU/mL) or AUC values in insulin observed after ingestion of the supplements .Results from the present study indicate that glucose and insulin concentrations peaked 30-min following ingestion of CHO/PRO and then proceeded to decline for 120-min. This finding is of interest from a nutrient delivery and timing standpoint in that it has been suggested that athletes should ingest CHO and PRO within two hours following intense exercise in order to optimize the hormonal effects of intense exercise and recovery . It has It is also interesting to note that glucose values in the H group stayed above baseline throughout recovery while values fell below baseline in the S and M groups. Consequently, this form of CHO may help maintain glucose levels and prevent incidents of hypoglycemia that some individuals may experience when ingesting large amounts of CHO and PRO. Although we did not measure glycogen uptake at the muscle, previous research has shown that ingesting CHO with higher GI following exercise promotes a more rapid resynthesis of muscle glycogen ,30,41,42Post-exercise ingestion of PRO and amino acids have been reported to stimulate PRO synthesis ,16,19,26Finally, research has shown that intense exercise causes an acute immuno-suppression for several hours after exercise. For this reason, a number of nutritional countermeasures including CHO and PRO have been proposed to lessen the immunosuppressive effects of intense exercise ,36. In tIn conclusion, CHO and PRO ingestion following exercise significantly influences glucose and insulin responses without significantly altering markers of anabolism, catabolism or immunity during the first two hours of recovery. Although ingesting honey as the source of CHO with PRO tended to maintain blood glucose levels to a greater degree, no significant differences were observed among the types of CHO ingested in terms of insulin response to supplementation. These findings suggest that each of these types of CHO can serve as effective sources of CHO to ingest with PRO following intense resistance-exercise in an attempt to optimize CHO availability as well as post-exercise anabolism.This study was funded by the National Honey Board  under the auspices of the United States Department of Agriculture (USDA).RBK: Obtained grant, served as PI of study, data analysis, manuscript preparationCE: Data analysis and manuscript preparationJL: Study coordinator and data collectionCR: Lab director, data collectionMG: Data collection and analysisPC: Medical supervision and data collectionALA: Research design, assisted in obtaining grant funding, data analysis


Benzbromarone, a uricosuric drug, reportedly causes hepatic hypertrophy accompanied by proliferation of peroxisomes in rats. To elucidate the mechanisms underlying induction of peroxisome proliferation by benzbromarone, we examined binding affinity for peroxisome proliferator-activated receptor  Recently, urate transporter 1 (URAT1) was identifiedin the luminal membrane of the proximal renal tubule, and the mechanism ofaction for benzbromarone was identified as suppression of uric acid resorptionvia inhibition of URAT1 . Clofibrα and PPARγ, and upregulates expression of eachprotein [α and PPARγ was reexamined in comparison with a representative activator of each receptor. In addition,regarding the mechanism of gene expression induced by benzbromarone, bindingbetween PPARs and peroxisome proliferator-responsive element (PPRE), and proteinexpressions of lipoprotein lipase (LPL), and acyl-CoA synthetase (ACS) encodedby the target gene of PPAR were examined.We have previously demonstrated that benzbromarone has affinity for PPARprotein . In thisBased on the findings, hepatic hypertrophy due to benzbromarone and the influence ofbenzbromarone on lipid metabolism are discussed.Benzbromarone, an active pharmaceutical ingredient of Urinorm marketed by Torii PharmaceuticalCo., Ltd. , was used as a test compound in this study. Troglitazone, pioglitazone and fenofibric acid were synthesized at the Central Research Pharmaceutical Institute of Japan Tobacco Inc. . Clofibric acid was purchased from Sigma-Aldrich Japan ,nuclear and cytoplasmic extraction reagents from Pierce , and a nucleic acid labeling kit fromMolecular Probes .μg/mL streptomycin at 37°C in a humidified 95% air and 5% CO2 atmosphere.NIH/3T3 cells and human kidney 293T cells were maintained in D-MEM containing 10% fetalbovine serum, 50 U/mL penicillin and 50 γ or the PPARα ligand-binding domain and GAL4 DNA-binding domain, together with a receptor plasmid containing the GAL4-responsive promoter driving expression of luciferase. After 6 hours, cells were givenfresh D-MEM containing test compounds at final concentrations of 0.001–100 μM, and were cultured for an additional 2 days.Benzbromarone, clofibric acid, fenofibric acid, troglitazone, and pioglitazonewere suspended in 0.1% DMSO solution. After cells had been lysed, luciferaseactivity was determined using a CT-9000D luminometer .NIH/3T3 cells . Oligonucleotides were labeledwith Alexa Flour 488 using a ULYSIS Nucleic Acid Labeling Kit (MolecularProbes). Labeled oligonucleotides (500 ng) were added to nuclear extracts. Thebinding assay buffer comprised 20 mM HEPES (pH 7.5) containing 40 mM KCl, 5% glycerol, and 1 μg dl-dC. Assay was performed at room temperature for 10 minutes at a final volume of 25 μL. Products of the binding reaction wereseparated on 5% polyacrylamide gel run in Tris-borate-EDTA at room temperatureat 100 V for 60 minutes. Gels were visualized using a Fluorimager SI . This test was performed 6 times.First, following addition of benzbromarone at a final concentration of 1 μM, and cells were cultured for 24 hours.Western blots for LPL and ACS proteins were then performed according toestablished procedures. Briefly, after cells (108 cells/well) had beensuspended in lysis buffer , aliquots of the cell suspension were obtained by repetitive pipettingand lysed by incubation at 4°C for 15 minutes. Lysed cells were centrifuged at400×g for 10 minutes, and the supernatant was separated by SDS-polyacrylamide gel electrophoresis in 10% acrylamide gel. The protein was transferred onto a nitrocellulose membrane and blocked for 60 minutes. The membrane was then incubated with anti-ACS  or anti-LPL  antibodies. After washing, the membrane was incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibodies . Chemiluminescence of bound antibodies was detected using a kit . Protein contents were determined by the generally established method of Lowry using BSA as a standard. This test was performed 4 times.Benzbromarone was added to 293T cells to achieve a final concentration of 1 or 10 α andPPARγ was examined by reporter gene assay using transcriptional activity of the PPAR target gene as a marker, in comparison with those of clofibric acid, fenofibric acid, troglitazone, andpioglitazone  and thiazolidine-class antidiabetic drugs , by comparisonto these PPAR activators.μM [Considering that ely 5 μM , which i


The Wong-Kilbourne derivative of Chang conjunctiva-derived cell line has been widely used for toxicological and functional in vitro studies on the ocular surface. The common reserve to this cell line is the reported contamination with HeLa cells. Thus, the IOBA-NHC spontaneously immortalized conjunctival epithelial cell line has been recently developed and did not show other cell type contamination. Our purpose was to determine whether both cell lines would be equally suitable for in vitro toxicological studies. Therefore, we compared in these two cell types the toxic effects of the preservative, benzalkonium chloride (BAC); its toxicity has been often reported on conjunctival in vivo and in vitro models.2DCF-DA], hydroethidine, and Yopro-1), flow cytometry (Annexin V/7-AAD and DNA content tests), and standard immunofluorescence stainings. Cells were exposed to five concentrations of BAC  for two incubation times: 15 min of treatment and 15 min of treatment followed by 24 h of cell recovery in complete medium.The necrotic, apoptotic, and oxidative effects of BAC were evaluated on Chang and IOBA-NHC cell lines using microplate cytofluorometry tests (neutral red, 2,7- dichlorofluorescein diacetate dye [HAll parameters of toxicity increased in a BAC dose-dependent manner on both cell lines.The comparison of BAC toxicity on both cell lines supported the use of IOBA-NHC and Chang cells for toxicological in vitro studies. Drawbacks of both cell lines have to be known and considered in studies performed on these cell lines. One of the best ways to investigate the ocular surface, especially the conjunctival epithelium, is theoretically to get human conjunctival biopsies that provide epithelial, stromal, inflammatory, and goblet cells ex vivo . However2 at 37 °C) in DMEM/F12 supplemented with 1 µg/ml bovine pancreas insulin, 2 ng/ml mouse epidermal growth factor, 0.1 µg/ml cholera toxin, 5 µg/ml hydrocortisone, 10% fetal bovine serum (FBS), 50 UI/ml penicillin, and 50 UI/ml streptomycin, as previously described [IOBA-NHC cells were cultured under standard conditions (humidified atmosphere of 5% COescribed .2 at 37 °C) in Dulbecco’s minimum essential medium supplemented with 10% fetal bovine serum, 1% glutamine, 50 UI/ml penicillin, and 50 UI/ml streptomycin.The Wong-Kilbourne derivative of Chang conjunctival cells  were cultured under standard conditions  for microtitration analysis , in 12 well culture plates  for flow cytometric analysis , and on slides  for standard immunofluorescence experiments. Cultures were kept at 37 °C for 24 h. Subconfluent cells (culture surface covering nearly 70%) were then exposed to the different BAC concentrations.−2%, 5.10−3%, 10−3%, 10−4%, and 10−5%) were analyzed. Indeed, these concentrations are equivalent to or lower than the concentrations of most available eye-drops. Concentrations of 10−1% and higher were not tested as they are known to be excessively toxic on conjunctival cells in vitro, inducing cell lysis immediately after treatment [Benzalkonium chloride was dissolved in phosphate buffered saline (PBS). Five different concentrations of BAC  [2.-) production  [2X7 cell death receptor activation [2DCF-DA, and Yopro-1 results were expressed using a ratio of the results of these tests to those of the neutral red test to correlate them to cellular viability.Microplate cytofluorometry was performed on the Saphire Microplate reader . Four different assays were used according to previously validated methods ,13-21. Aene, OR) , and theene, OR) . The Yoptivation -21. In aAnnexin/V-7AAD staining and DNA content analysis are two apoptotic tests that were assessed by flow cytometry on a FC 500-CXP flow cytometer  equipped with an argon laser emitting at 488 nm ,23. Anne1 peak flow cytometric analysis on a FL4-fluorescence histogram in a linear mode to characterize the late apoptosis process. After 15 min of BAC treatment followed by 24 h of cell recovery in complete medium, cells were collected using EDTA as previously described and fixed with 0.5% paraformaldehyde (PFA) fixative in PBS at 4 °C for 24 h. After the 24 h fixation, samples were washed with cold PBS, permeabilized in 0.1% saponin, stained with 50 µg/ml propidium iodide , and analyzed on the flow cytometer. The sub-G1 region was determined by a cursor defined in the controls and excluding the debris as described previously [Alteration of DNA content was measured through the sub-Geviously .Standard immunofluorescence staining under a confocal epifluorescence microscope  was performed to assess morphologic patterns of cells. Cells were cultured on chamber slides  and incubated with the different BAC concentrations for 15 min. They were then washed with PBS and fixed for 15 min with 4% PFA in PBS at 20 °C. Fluorescent phalloidin  was then added to detect F-actin. After 30 min of incubation with phalloidin, cells were washed in PBS before propidium iodide staining and microscopy examination.t-test.Statistical comparisons were performed with Sigma Stat 2.0 . For the microplate cytofluorometry data, significance was assessed using one-way analysis of variance (ANOVA) followed by the Dunnett test. The flow cytometry data were analyzed using one-way ANOVA followed by the Bonferroni −2%  followed by BAC 5.10−3%  then BAC 10−3%, 10−4%, and 10−5% . On IOBA-NHC cells, membrane integrity results were 36% of the control with BAC 10−2% (p<0.001), 56% with BAC 5.10−3% (p<0.001), 95% with BAC 10−3% (NS), 109% with BAC 10−4% (p<0.001), and 106% with BAC 10−5% (p<0.05).After a 15 min incubation time, the membrane integrity decreased in a concentration-dependent manner on the two cell lines . Thus, o−4% and 10−5%. With BAC 10−3%, cellular viability increased on IOBA-NHC cells and decreased on Chang cells while with BAC 5.10−3% and 10−2%, cellular viability decreased on both cell lines.After 24 h of cell recovery , cellula−2%,  followed by BAC 5.10−3%  then BAC 10−3%, 10−4%, and 10−5% . On IOBA-NHC cells, the highest ratio was also obtained with BAC 10−2%  followed by BAC 5.10−3% , BAC 10−3% , 10−4% , and 10−5% .After a 15-min exposure time, the highest ROS production was obtained with the highest BAC concentrations and varied in a concentration-dependent manner . Indeed,−3% and lower whereas ROS production increased with BAC 5.10−3% and BAC 10−2%.After 24 h of cell recovery  in both −2%  followed by BAC 5.10−3% , but results were not different from the control at the three other concentrations . On IOBA-NHC cells, increased ratios were obtained with BAC 10−2%  and BAC 5.10−3% . BAC 10−3% was not different from the control , and BAC 10−4% and 10−5% significantly decreased superoxide anion production .After a 15 min exposure time, the highest superoxide anion production was obtained with the highest BAC concentrations and similarly increased in a concentration dependent manner . On Chan−3%, and decreased with the other four BAC concentrations.After 24 h of cell recovery , superoxAfter 15 min of cell exposure to the different concentrations of BAC, we observed that the Yopro-1/neutral red ratio consistently increased with BAC concentration .−2%, 5.10−3%, 10−3%, 10−4%, and 10−5%, respectively. On IOBA-NHC cells, lower ratios were obtained, 7.29 (p<0.001), 4.05 (p<0.001), 1.64 (p<0.001), 1.32 (p<0.05), and 1.29 (p=NS) for BAC 10−2%, 5.10−3%, 10−3%, 10−4%, and 10−5%, respectively.On Chang cells, ratios were 9.04, 8, 2.17, 2.18, and 2.19  for BAC 10After 24 h of cell recovery , the rat−2%, 5.10−3%, 10−3%, 10−4%, and 10−5%, respectively. Similar results were obtained on IOBA-NHC cells, with percentages of 92.9% (p<0.001), 74.1% (p<0.001), 27.5% (p<0.001), 17.1% (p=NS), and 17.4% (p=NS) for BAC 10−2%, 5.10−3%, 10−3%, 10−4%, and 10−5%, respectively.For the total toxic effect evaluation (apoptotic and necrotic cells), results were expressed in percentage of cells engaged in a cell death process. With this global parameter, the same toxicity profiles were obtained on the two cell lines with BAC concentration-dependent cell toxicity . On Chan−5%  and with BAC at 10−4% . Conversely, when concerning the early apoptotic and necrotic cell populations in Chang cells or the early apoptotic, late apoptotic, and necrotic cell populations in IOBA cells, no statistically significant difference was shown between these same BAC concentrations and PBS. With BAC 10−3%, the late apoptotic cell population was significantly increased in both Chang cells  and IOBA-NHC cells  whereas the early apoptotic and the necrotic cell populations were not significantly increased in both cell lines. In addition, no statistically significant difference was found between the two cell lines in the late apoptotic cell population with BAC 10−3%. With BAC 5.10−3%, on Chang cells, the percentages of late apoptotic cells and necrotic cells in Chang cells were 21.9% and 59.7% (p<0.05 versus control), respectively. On IOBA-NHC cells, there was a higher percentage of late apoptotic cells  and a lower percentage of necrotic cells  with BAC at 5.10−3%.With the Annexin/V-7 AAD test, we also analyzed the early apoptotic, late apoptotic, and necrotic cell populations , discrim1 events when compared to the total cell population after 15 min of treatment followed by 24 h cell recovery in normal culture medium , 69.9% (p<0.001), 28.1% (p<0.05), 17.6% (p=NS), and 16.6% (p=NS), respectively, for the same BAC concentrations.Results were expressed in the percentage of sub-Ge medium . The sam−2%  production, cell toxicity increased after the 24 h recovery period on Chang cells but tended to decrease on IOBA-NHC cells except with a BAC concentration at 5.10−3%. Conversely, using the Yopro-1 test, we observed that the Yopro-1/neutral red ratio consistently decreased on Chang cells unlike on IOBA-NHC cells after 24 h of cell recovery. Thus, further experiments could be useful to study the cell characteristics potentially involved in these different cell behaviors such as the oxidative or anti-oxidative status of each cell line or the P2X7 cell death receptor expression after a single 15 min incubation time and after the 24 h recovery period.In this study, all parameters of toxicity measured increased in a BAC dose-dependent manner on both cell lines. Indeed, the highest toxicity was obtained with the highest BAC concentrations on both Chang and IOBA-NHC cells after a 15 min exposition time and after 24 h of cell recovery. In addition, the BAC threshold concentration observed was BAC at 5.10n et al.  on Chang−5% and 10−4% in Chang cells whereas on IOBA-NHC cells, the toxicity was only shown with BAC at 10−3% and higher. With BAC at 5.10−3%, most of IOBA-NHC cells were in a late apoptotic process whereas most of Chang cells were necrotic, underlining the higher sensitivity of Chang cells. Several factors could explain thethese differences: the mitochondrial cells status, the expression or activation of cell death receptors , or the factors or enzymes implicated in the oxidative stress  or in the apoptotic process . Another parameter that could explain these differences is the supplementation of IOBA-NHC cell culture medium with several components like mouse epidermal growth factor and hydrocortisone that could enhance the resistance of cells against BAC. In addition, with the neutral red test, cellular viability was significantly increased in IOBA-NHC cells with BAC at 10−5% and 10−4% after 15 min of cell treatment but mostly after 24 h of cell recovery. This effect could be explained by a higher cell growth of this cell line compared to Chang cells, but further experiments are required to confirm this hypothesis.In the present study, other small differences have been found between the two conjunctival cell lines. Indeed, BAC toxicity seemed to be higher on the Chang cell line than on the IOBA-NHC cell line, suggesting a higher sensitivity in Chang cells toward the toxic effects of BAC. With the sensitive Annexin/V-7AAD apoptosis test, a BAC toxicity (late apoptosis) was found with BAC at 10In the present study, the Wong-Kilbourne derivative of Chang conjunctival cells seemed to have a high sensitivity to BAC. These data suggest that the HeLa cell contamination to Wong-Kilbourne derivative of Chang conjunctival cells does not affect the usefulness of the cell line, supporting the use of Chang cells for toxicological, in vitro studies. Thus, as it has been previously reported, mild toxic effects of preservative or other potentially toxic agents can be accurately screened using this cell line ,27,28,39In conclusion, this present study demonstrates that the comparison of BAC toxicity on both Chang and IOBA-NHC cell lines supported the use of IOBA-NHC cells for toxicological studies in vitro and validated previous studies performed on the Chang cells despite their reported contamination with HeLa cells. Thus, our findings confirm that both cell lines are useful for toxicological in vitro studies as their respective disadvantages are known and discussed. Particularly, IOBA-NHC cells, which showed no other cell type contamination, are cultured in a medium that is supplemented with epidermal growth factor (EGF), hydrocortisone, and cholera toxin, factors which may be potentially implicated in the cell resistance against a toxic agent like BAC. Further studies could be performed to determine whether the deprivation of each factor could modify cell functional behavior and morphologic characteristics. Other factors could influence cell sensitivity to xenobiotics like the oxidative or mitochondrial status of tested cells and the expression of death receptors that could be characterized in further studies. Moreover, comparison of the characteristics of both cell lines will deserve further interest to complete the validation of the usefulness of these cell lines for functional studies.


Maternal infection during pregnancy has been associated with increased incidence of schizophrenia in the adult offspring. Mechanistically, this has been partially attributed to neurodevelopmental disruption of the dopamine neurons, as a consequence of exacerbated maternal immunity. In the present study we sought to target hypoferremia, a cytokine-induced reduction of serum non-heme iron, which is common to all types of infections. Adequate iron supply to the fetus is fundamental for the development of the mesencephalic dopamine neurons and disruption of this following maternal infection can affect the offspring's dopamine function. Using a rat model of localized injury induced by turpentine, which triggers the innate immune response and inflammation, we investigated the effects of maternal iron supplementation on the offspring's dopamine function by assessing behavioral responses to acute and repeated administration of the dopamine indirect agonist, amphetamine. In addition we measured protein levels of tyrosine hydroxylase, and tissue levels of dopamine and its metabolites, in ventral tegmental area, susbtantia nigra, nucleus accumbens, dorsal striatum and medial prefrontal cortex. Offspring of turpentine-treated mothers exhibited greater responses to a single amphetamine injection and enhanced behavioral sensitization following repeated exposure to this drug, when compared to control offspring. These behavioral changes were accompanied by increased baseline levels of tyrosine hydroxylase, dopamine and its metabolites, selectively in the nucleus accumbens. Both, the behavioral and neurochemical changes were prevented by maternal iron supplementation. Localized prenatal inflammation induced a deregulation in iron homeostasis, which resulted in fundamental alterations in dopamine function and behavioral alterations in the adult offspring. These changes are characteristic of schizophrenia symptoms in humans. Environmental factors, combined with genetic predisposition, are now recognized as key events underlying a number of psychiatric disorders of neurodevelopmental origin, including schizophrenia. Of the environmental events, maternal infection during critical stages of human gestation has been associated with increased incidence of schizophrenia in the adult progeny We hypothesized that inflammation-induced hypoferremia causes a disruption of fetal brain development, which leads to functional defects in adulthood, synonymous with psychiatric disorders such as schizophrenia. The goal of this study was, therefore, to investigate whether changes in iron traffic induced by maternal inflammation would have an impact on DA function and DA-related behaviors in the adult offspring. To this end, we conducted studies using a rat model of localized injury and inflammation induced by an intramuscular (i.m.) injection of turpentine (TURP). In contrast to the more commonly used models of systemic bacterial [lipopolysaccharide (LPS)] or viral (poly I:C) infection, TURP remains localized at the injection site  = 7.74, p<0.001, simple effect of treatment at 10 h: F = 37.82, p<0.0001). Serum pro- [interleukin (IL)-6)] and anti- [IL-1 receptor antagonist (IL-1ra)] inflammatory cytokines were significantly induced at the same time point (10 h) after TURP injection and no longer detectable by 24 h  = 3.98, p = 0.004, simple effect of treatment at 10 h F = 17.5, p<0.0001; IL-1ra: treatment by time interaction F = 11.04, p<0.0001, simple effect of treatment at 10 h F = 52.2, p<0.0001).In order to characterize the maternal inflammatory response to TURP and the alterations that this may induce in systemic iron trafficking, we determined the kinetics of several inflammatory markers and maternal serum iron levels at GD 15. TURP induced a significant increase in core body temperature at 10 hours, with a 1.9°C difference in comparison to the saline (SAL) group and returned to basal 24 h after injection  = 14.10, p<0.0001; simple effect of treatment at 10 h, F = 12.34, p = 0.0007; at 24 h, F = 69.26, p<0.0001), returning to control levels at 48 h (GD 17) after treatment. No further differences in non-heme iron levels were observed between TURP and control groups. However, there was a reduction in the serum levels of iron with the progress of pregnancy towards term, with non-heme iron levels at GD 19 becoming significantly lower than those at GD 15, 16, 17 and 18  = 16.36, p<0.0001, 96 h vs. 0, 24, 48 or 72 h Fisher's LSDs p< 0.05). This decrease in maternal serum non-heme iron towards term is in fact normally observed, as iron is used to sustain increased requirement for this nutrient due to expansion of the maternal erythrocyte mass and the high demand of the growing fetus TURP treatment dramatically reduced serum non-heme iron levels, 10 and 24 h after injection ((12) = 5.27, p = 0.0002) and zip14 mRNA by 5.5-fold ((12) = 4.48, p = 0.0007). Zip14 is a dual iron/zinc importer involved in the cellular uptake of iron (12) = 7.6, p<0.0001), indicating the activation of the JAK/STAT signaling pathway, most likely by IL-6 TURP's effect on circulating iron levels was accompanied by an induction of hepatic expression levels of HAMP mRNA by 25-fold  = 7.85, p = 0.01; simple effect of maternal treatment for VEH supplemented animals, F = 24.40, p<0.0001), supporting the effect seen by analyzing raw serum non-heme iron levels  = 0.95, p = 0.33), suggesting a blockade of the TURP effect on serum iron levels when mothers were over-loaded with this micronutrient. Basal Tf saturation among the iron supplemented animals was significantly lower than VEH mothers  = 4.91, p = 0.0004, simple effect of supplementation at 0 h, F = 10.8, p = 0.0013), which may be due to the induction of hepatic HAMP mRNA expression, as observed in both SAL-IRON and TURP-IRON groups at GD 19  = 5.89, p = 0.044; SAL-IRON or TURP-IRON vs. SAL-VEH or TURP VEH Fisher's LSDs, p<0.05). Accordingly, there was an accumulation of non-heme iron in the maternal liver of iron-supplemented dams at this same time point  = 23.18, p<0.0001; SAL-IRON or TURP-IRON vs. SAL-VEH or TURP VEH Fisher's LSDs, p<0.0001), in the absence of all the other TURP-induced effects, which returned to basal/control levels [maternal serum non-heme iron  = 4.36, p = 0.007; SAL-VEH vs. TURP-VEH Fisher's LSD, p<0.05), and this effect was reversed by iron supplementation in TURP-IRON treated dams (p<0.05). HAMP mRNA expression levels in this organ paralleled the effects seen in iron content, with a reduction induced by TURP treatment  = 6.37, p = 0.0029; non significant SAL-VEH vs. TURP-VEH Fisher's LSD, p = 0.07) and a recovery of HAMP mRNA expression in the TURP-IRON group, to levels significantly greater than those of SAL-VEH (p<0.05).We performed fetal iron content and HAMP mRNA expression measurements 96 h after TURP treatment. This time point was chosen since the acute inflammatory responses to TURP  had returned to basal/control levels, enabling us to study the extended effects of the inflammatory challenge on fetal tissue in the absence of the maternal immune responses. In the placenta, non-heme iron content was significantly reduced in the TURP-VEH treated group  = 0.71, p = 0.55). However, fetal hepatic HAMP mRNA levels were significantly increased in the fetuses from TURP- and SAL-IRON treated mothers  = 4.17, p = 0.018; SAL-VEH vs. TURP-VEH or SAL-IRON Fisher's LSD, p<0.05), whereas in fetal livers from TURP-IRON treated mothers, HAMP mRNA levels were back to control levels (p<0.05).There were no effects on fetal liver iron content  = 2.96, p = 0.038; TURP-IRON vs. SAL-VEH or TURP-VEH Fisher's LSDs, p<0.05). However, there were no effects on HAMP mRNA expression  = 0.46, p = 0.71).In the fetal brain, non-heme iron content was significantly reduced in the fetuses of TURP-IRON treated dams, compared to the fetal brains from SAL-VEH and TURP-VEH treated mothers , suggesting that neither TURP and/or iron supplementation induced fetal mortality nor gross physical changes at birth. = 4.84, p = 0.032; simple effect of treatment in the VEH condition, F = 6.96, p = 0.01). In contrast, there was no difference in AMPH-induced locomotion between the offspring of TURP and SAL-treated, iron-supplemented mothers  = 0.22, p = 0.64), which exhibited locomotor responses to AMPH statistically similar to those of the SAL-VEH group  = 2.32, p = 0.13). During days 1 and 2, iron supplemented animals presented a trend for enhanced response to AMPH in comparison to SAL-VEH, which disappeared from day 3 onwards; this effect did not reach statistical significance.In the adult offspring, locomotion in basal conditions or in response to amphetamine  or saline injection was determined. AMPH or saline were administered to animals of each experimental group for 5 consecutive days. No effects were observed in basal locomotion  or in re = 63.9, p<0.0001). However, among the AMPH pre-treated animals, offspring of dams exposed to TURP-VEH showed a significantly greater sensitized response than the offspring of the SAL-VEH group  = 7.83, p = 0.006; simple effect of prenatal treatment for the AMPH pre-treated VEH animals, F = 10.39, p = 0.0016). In contrast, offspring of TURP-IRON treated dams were sensitized to AMPH similar to the SAL-VEH offspring  = 12.17, p = 0.0007). Intriguingly, iron supplementation alone during pregnancy, also resulted in enhanced behavioral sensitization in the adult offspring  = 9.94, p = 0.02; simple effect of treatment for the AMPH pre-treated IRON animals, F = 8.77, p = 0.0037).In the test for behavioral sensitization (7 days after last pre-treatment injection), all animals were challenged with a single injection of AMPH (1 mg/kg). As expected, all AMPH pre-treated rats exhibited sensitized response to the AMPH challenge in comparison to those pre-treated with saline  = 3.63, p = 0.028; TURP-VEH vs. SAL-VEH or TURP-IRON Fisher's LSDs, p<0.05). These changes were region specific, as levels of TH expression in the dorsal striatum  = 0.62, p = 0.60), medial prefrontal cortex  = 0.88, p = 0.46), ventral tegmental area  = 0.58, p = 0.63) and substantia nigra  = 0.04, p = 0.96) were not significantly altered by any prenatal treatment.We analyzed baseline tyrosine hydroxylase (TH) expression in DA rich regions by Western blotting . Consist = 3.83, p = 0.028; SAL-VEH vs. TURP-VEH Fisher's LSD, p<0.05), as well as those of DOPAC  = 18.27, p<0.0001; SAL-VEH vs. TURP-VEH Fisher's LSDs, p<0.05) and HVA  = 3.83, p = 0.041, SAL-VEH vs. TURP-VEH Fisher's LSDs, p<0.05). Intriguingly, DA levels in the SAL-IRON and TURP-IRON groups were also increased (p<0.05). As for TH, effects of prenatal treatments appeared to be specific to the NAcc since neither dSTR  = 3.26, p = 0.043; SAL-VEH vs. SAL-IRON Fisher's LSD, p<0.05).We also measured tissue content of DA, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in DA terminal regions . Consisther dSTR  nor mPFCher dSTR  DA and HIn the current study we provided the first evidence, to our knowledge, that localized maternal inflammation during gestation renders adult offspring significantly more sensitive to the locomotor activating effects of a single AMPH injection, and to the behavioral plasticity following repeated exposure to this drug. In addition, we demonstrated increased baseline expression of TH and tissue levels of DA, DOPAC and HVA, which were specific to the NAcc. The fact that TURP does not enter the circulation suggests that the effects observed in the offspring in the current study, are most likely due to secondary downstream mediators and not to direct effects on the fetal compartment, as reported for poly I:C We demonstrated that maternal iron supplementation, which counteracts inflammation-induced hypoferremia, effectively prevented the prenatal TURP-induced increased sensitivity to the behavioral effects of single and repeated AMPH administration in the adult offspring. These results suggest that maternal hypoferremia, induced by the TURP injection, causes long-lasting behavioral alterations in the offspring. Remarkably, prenatal TURP-induced increase in NAcc TH expression was also completely abrogated by maternal iron supplementation.Increased levels of TH and DA, restricted to the NAcc, have also been reported by others in the offspring of mothers treated with poly I:C Increased NAcc TH, which is consistent with augmented basal NAcc DA concentration, may result from either a change in expression level per neuronal terminal, or from structural reorganization within the mesolimbic circuitry, the latter resulting in axonal sprouting of NAcc DA terminals. Prenatal inflammation may therefore lead to profound presynaptic alterations in NAcc DA synthesis. However, additional effects on DA receptors, transporter Interestingly, iron supplementation alone (i.e. SAL-IRON group) induced several behavioral and biochemical changes in the offspring, which resembled the effects of prenatal inflammation . SAL-IRON group also exhibited a greater AMPH-induced stereotypy during the pre-treatment phase (data not shown) and increased basal levels of DOPAC in the dSTR. This surprising finding is most likely due to iron overload in iron sufficient animals Given that maternal circulation constitutes the only source of iron to the developing fetus The changes in iron levels in the placenta were very closely paralleled by the levels of HAMP expression, which may be merely responding to the tissue's fluctuation in iron concentration Iron levels in the fetal brains taken from all four treatment groups did not show any alteration, other than a small but significant reduction in TURP-IRON group. This latter observation is rather puzzling and it possibly reflects a lower than normal supply of iron in the TURP-IRON group. The latter effect can be mediated by the increased placental HAMP in the TURP-IRON group, which would lead to iron trapping within this organ, at the expense of the fetal brain, similar to the effects of maternal hepatic HAMP on maternal liver iron content Hypoferremia has long been recognized as one of the main components of the host's response to infection/inflammation, and the mechanisms involved in the induction of this process are fairly well understood. The strongest evidence to date suggests that the pro-inflammatory cytokine IL-6, which is readily detectable in the circulation of infected individuals regardless of the pathogenic trigger, is the main circulating mediator of hepatic HAMP mRNA expression Our results clearly demonstrate that a localized inflammatory insult during gestation has profound affects on DA function, which may be relevant for schizophrenia, where increased striatal DA is proposed to underlie the so-called positive symptoms of the disorder st trimester of human pregnancy Time pregnant primiparous Sprague-Dawley rats  were used in all experiments. On GD 7 or 8 animals were individually housed in a controlled environment at an ambient temperature of 21±1°C on 12:12 h light-dark cycle (lights on at 0800 hours) with free access to food and water. Only male offspring were used, which were housed under the same environmental conditions. Mothers were treated with the inflammatory stimulus at GD 15. This day of pregnancy was used since it has been proposed to be roughly equivalent to the late 1Ethics statement. Experimental procedures were approved by the Animal Care Committee from the Douglas Mental Health University Institute and McGill University pursuant of the Canadian Council of Animal Care . All efforts were made to minimize the number of animals used.Pregnant dams were handled daily from GD 11 onwards and habituated to a rectal probe to determine core temperature . Some animals (13 dams) received an intraperitoneal (i.p.) injection of 10 mg/kg of the aqueous complex of poly-nuclear iron (III)-hydroxide in sucrose  or an equivalent volume of vehicle  at GD 13 and 14. Iron was administered to the dams from GD 15 until GD 18 at larger doses . At GD 15 basal core temperature was recorded and a small sample of tail blood was collected to determine basal levels of cytokines and serum iron. Subsequently, a maternal inflammatory response was elicited by injecting intramuscularly with 100 µL of purified TURP ; control dams received an equivalent volume of saline. All injections were administered between 0900 and 1000 hours. Using a rectal probe core body temperature was measured at 8, 10, 24, 48 and 72 hours after i.m. treatment and tail blood collected at each time point. The animals were then sacrificed at GD 19 (96 hours after the i.m. treatment) with a lethal dose of pentobarbital sodium  and a final blood sample was collected via cardiac puncture. Maternal liver, placentas, fetal livers and brains were excised and immersed in 2-methylbutane  and chilled with dry ice. Serum cytokine, iron and Tf saturation levels were determined, as well as maternal liver, placenta, fetal liver and brain tissue iron levels.In a separate experiment, SAL and TURP treated dams  were sacrificed 10–11 hours after i.m. treatment, which represents the peak of fever and cytokine responses To determine the effects of prenatal TURP and iron supplementation on DA function and related behaviors in the adult offspring, pregnant dams (6–12 per group) were treated as described above, with the exception that tail blood collection was not performed. The male offspring from each mother were marked, weighed and cross-fostered with surrogate dams in mixed litters We measured locomotor responses to either single or repeated injections of d-amphetamine sulphate salt  in adult male offspring, using our well-established protocol All blood samples were allowed to cloth at room temperature for 1 hour, spun at 4000 rpm for 20 min at 4°C to obtain serum and stored at −80°C until used. Maternal serum samples were analyzed for IL-6 and IL-1ra  high-purity water. One volume of protein-precipitation solution (1 N HCl and 10% (v/v) trichloroacetic acid in high purity water) was then added to the samples, blank and iron standards and incubated for 1 hour at 95°C. The samples were then allowed to cool at room temperature for 2 min, vortex mixed and centrifuged at 10 000×Total RNA was extracted and reverse transcribed from maternal livers, placenta, fetal liver and brain as described previously We explored biochemical markers of DA neurons, including protein expression of TH and tissue levels of DA and its metabolites, DOPAC and HVA. Brains collected from adult male offspring (P 60–62) by decapitation were immersed in 2-methylbutane  and chilled with dry ice. 300 µm thick cryostat sections were obtained. There were no behavioral differences between the animals born to mothers supplemented with either SAL or sucrose. Thus, to minimize the number of animals used, only SAL-SAL and TURP-SAL groups were included. Bilateral punches from the VTA, SN, NAcc (including core and shell), dSTR and mPFC (including cingulated areas 1 and 2) were excised using our previously described procedures Western blots were conducted as previously described HPLC analysis was conducted as we recently reported p values<0.05 values were deemed significant.All data were analyzed as raw values, except for HPLC results, where the data were analyzed as percentage of the average value for the SAL-VEH group, due to large inter-assay variability between two different batches of samples assayed. All data are presented as mean values ± s.e.m. Maternal fever, serum cytokines and iron: data were analyzed using a repeated measures 2-way ANOVA, with time as a within-group variable and pre-natal treatment (SAL vs. TURP) as a between-groups variable.t-tests to compare data from the SAL-VEH and TURP-VEH groups.Maternal liver PCR data: data from the 10–11 hours experiment were analyzed using two-tailed Student's Maternal serum Tf saturation: data were analyzed using a 3-way ANOVA with prenatal treatment (SAL vs. TURP) and maternal supplementation (VEH vs. IRON) used as between-groups variables in all cases and time point used as a within-group variable. When the interactions of main effects were significant, analysis was followed by performing simple-effects ANOVA and Fisher's LSD post hoc tests.Tissue iron content, HAMP mRNA levels (96 h time point), western blotting and HPLC: data were analyzed using 1-way ANOVA, with maternal treatment (SAL-VEH vs. TURP-VEH vs. SAL-IRON vs. TURP-IRON) as between-groups variable and furthered with Fisher's LSD post hoc tests when ANOVA was significant.Offspring's basal and AMPH-induced locomotion: data were analyzed using 3-way ANOVAs, with prenatal treatment (SAL vs. TURP) and maternal supplementation (VEH vs. IRON) as between-groups variables and time as a within-group variable. When interactions were significant, analyses were followed by simple-effects ANOVA and Fisher's LSD post hoc tests.AMPH sensitization test: data were analyzed using a 3-way ANOVA, with prenatal treatment (SAL vs. TURP), maternal supplementation (VEH vs. IRON) and pre-treatment  used as a between-groups variables, followed by simple-effects ANOVA and Fisher's LSD post hoc tests when interactions were significant.Figure S1Inflammatory response remained intact in iron supplemented mothers. (A) Febrile response followed the same kinetics in vehicle and iron supplemented mothers, peaking at 10 h after TURP injection and returning to baseline 24 h later. Basal temperature was not affected by iron supplementation. (B and C) Serum IL-6 and IL-1ra levels followed the same kinetics in the TURP-IRON group compared to the TURP-VEH group.(4.70 MB TIF)Click here for additional data file.