High Pharmacological Doses Research Articles

Flattening of circadian glucocorticoid oscillations drives acute hyperinsulinemia and adipocyte hypertrophy.

Disruption of circadian glucocorticoid oscillations in Cushing's disease and chronic stress results in obesity and adipocyte hypertrophy, which is believed to be a main source of the harmful effects of obesity. Here, we recapitulate stress due to jet lag or work-life imbalances by flattening glucocorticoid oscillations in mice. Within 3days, mice achieve a metabolic state with persistently high insulin, but surprisingly low glucose and fatty acids in the bloodstream, that precedes a more than 2-fold increase in brown and white adipose tissue mass within 3weeks. Transcriptomic and Cd36-knockout mouse analyses show that hyperinsulinemia-mediated de novo fatty acid synthesis and Cd36-mediated fatty acid uptake drive fat mass increases. Intriguingly, this mechanism by which glucocorticoid flattening causes acute hyperinsulinemia and adipocyte hypertrophy is unexpectedly beneficial in preventing high levels of circulating fatty acids and glucose for weeks, thus serving as a protective response to preserve metabolic health during chronic stress.

Vitamin B12 does not increase cell viability after hydrogen peroxide induced damage in mouse kidney proximal tubular cells and brain endothelial cells

Vitamin B12 (B12) is an essential co-factor for two enzymes in mammalian metabolism and can also act as a mimetic of superoxide dismutase (SOD) converting superoxide (O2•−) to hydrogen peroxide (H2O2). High oral dose B12 decreases renal O2•− and post-ischemia/reperfusion injury in mice and protects against damage induced by hypoxia/reperfusion in mouse kidney proximal tubular cells (BU.MPT). O2•− is unstable and rapidly converted to H2O2. H2O2 mediates oxidative stress associated with O2•−. Whether B12 protects against damage induced by H2O2 is unknown. Both BU.MPT cells and mouse brain endothelial cells (bEdn.3) were applied to test the effects of B12 on H2O2-induced cytotoxicity. Both types of cells were treated with different doses of H2O2 with or without different doses of B12. Cell viability was analyzed 24 h later. H2O2 caused cell death only at a very high dose, and high pharmacological dose of B12 did not prevent this detrimental effect in either cell type. In bEnd.3 cells, transcriptional levels of heme oxygenase-1 (HO-1) increased, while nuclear factor erythroid 2-related factor 2 (Nrf2) decreased by H2O2. The levels of transcripts were not affected by the B12 treatment. We conclude that the cytotoxic effects of exogenous H2O2 in BU.MPT and bEdn.3 cells are not prevented by B12.

Thyroid hormones in the regulation of brown adipose tissue thermogenesis.

A normal thyroid status is crucial for body temperature homeostasis, as thyroid hormone regulates both heat loss and conservation as well as heat production in the thermogenic tissues. Brown adipose tissue (BAT) is the major site of non-shivering thermogenesis and an important target of thyroid hormone action. Thyroid hormone not only regulates the tissue’s sensitivity to sympathetic stimulation by norepinephrine but also the expression of uncoupling protein 1, the key driver of BAT thermogenesis. Vice versa, sympathetic stimulation of BAT triggers the expression of deiodinase type II, an enzyme that enhances local thyroid hormone availability and signaling. This review summarizes the current knowledge on how thyroid hormone controls BAT thermogenesis, aiming to dissect the direct actions of the hormone in BAT and its indirect actions via the CNS, browning of white adipose tissue or heat loss over body surfaces. Of particular relevance is the apparent dose dependency of the observed effects, as we find that minor or moderate changes in thyroid hormone levels often have different effects as compared to high pharmacological doses. Moreover, we conclude that the more recent findings require a reevaluation of older studies, as key aspects such as heat loss or central BAT activation may not have received the necessary attention during the interpretation of these early findings. Finally, we provide a list of what we believe are the most relevant questions in the field that to date are still enigmatic and require further studies.

Amelioration of adrenaline induced oxidative gastrointestinal damages in rat by melatonin through SIRT1-NFκB and PGC1α-AMPKα cascades

Adrenaline at high pharmacological doses may lead to oxidative damages in diverse organs including gut. In this study, we attempt to elucidate the potentially protective effects of melatonin on gastrointestinal (GI) tissue damages induced by adrenaline. Rats were injected (s.c.) with different doses (0.125, 0.25 and 0.50 mg/kg) of adrenaline bitartrate (AD) for 15 days with or without melatonin (2.5, 5 and 10 mg/kg; orally). The results showed that adrenaline caused massive histological and ultra-structural GI injuries and melatonin (20 mg/kg) effectively protected these injuries. The protective mechanisms are related to the antioxidant and anti-inflammatory activities of melatonin indicated by increased glutathione levels and antioxidant enzymes as well as decreased oxidative stress markers and pro-inflammatory cytokines in GI tissues. The signal pathways of melatonin include up-regulating expression of Nrf2, SIRT1 and Bcl2, while down-regulating NFκB, TNFα and Bax. Melatonin also targeted mitochondrial energy homeostasis and biogenesis by up-regulating expression of PGC1α, AMPKα and SOD2 and reduced leakage of cytochrome c. The SIRT1-NFκB and PGC1α-AMPKα signal transduction pathways seem to play the central roles involving in melatonin’s protective effects on gastric damages induced by the high doses of adrenaline.

Pro- and Antioxidant Effects of Vitamin C in Cancer in correspondence to Its Dietary and Pharmacological Concentrations.

Vitamin C is an antioxidant that may scavenge reactive oxygen species preventing DNA damage and other effects important in cancer transformation. Dietary vitamin C from natural sources is taken with other compounds affecting its bioavailability and biological effects. High pharmacological doses of vitamin C may induce prooxidant effects, detrimental for cancer cells. An oxidized form of vitamin C, dehydroascorbate, is transported through glucose transporters, and cancer cells switch from oxidative phosphorylation to glycolysis in energy production so an excess of vitamin C may limit glucose transport and ATP production resulting in energetic crisis and cell death. Vitamin C may change the metabolomic and epigenetic profiles of cancer cells, and activation of ten-eleven translocation (TET) proteins and downregulation of pluripotency factors by the vitamin may eradicate cancer stem cells. Metastasis, the main reason of cancer-related deaths, requires breakage of anatomical barriers containing collagen, whose synthesis is promoted by vitamin C. Vitamin C induces degradation of hypoxia-inducible factor, HIF-1, essential for the survival of tumor cells in hypoxic conditions. Dietary vitamin C may stimulate the immune system through activation of NK and T cells and monocytes. Pharmacological doses of vitamin C may inhibit cancer transformation in several pathways, but further studies are needed to address both mechanistic and clinical aspects of this effect.

Synthesis and evaluation of two new candidate high-affinity full agonist PET radioligands for imaging 5-HT1B receptors

IntroductionThe serotonin 1B receptor subtype is of interest in the pathophysiology and treatment of depression, anxiety, and migraine. Over recent years 5-HT1B receptor binding in human brain has been examined with PET using radioligands that are partial but not full agonists. To explore how the intrinsic activity of a PET radioligand may affect imaging performance, two high-affinity full 5-HT1B receptor agonists (AZ11136118, 4; and AZ11895987, 5) were selected from a large compound library and radiolabeled for PET examination in non-human primates. Methods[11C]4 was obtained through Pd(0)-mediated insertion of [11C]carbon monoxide between prepared iodoarene and homochiral amine precursors. [11C]5 was obtained through N-11C-methylation of N-desmethyl precursor 6 with [11C]methyl triflate. [11C]4 and [11C]5 were studied with PET in rhesus or cynomolgus monkey. [11C]4 was studied with PET in mice and rats to measure brain uptake and specific binding. Ex-vivo experiments in rats were performed to identify whether there were radiometabolites in brain. Physiochemical parameters for [11C]4 (pKa, logD and conformational energetics) were evaluated. ResultsBoth [11C]4 and [11C]5 were successfully produced in high radiochemical purity and in adequate amounts for PET experiments. After intravenous injection of [11C]4, brain radioactivity peaked at a low level (0.2 SUV). Pretreatment with tariquidar, an inhibitor of the brain P-gp efflux transporter, increased brain exposure four-fold whereas pretreatment with a high pharmacological dose of the 5-HT1B antagonist, AR-A000002, had no effect on the binding. Ex-vivo experiments in rats showed no radiometabolites entering brain. [11C]5 also failed to enter monkey brain under baseline conditions. Conclusions[11C]4 and [11C]5 show too low brain uptake and specific binding to be useful PET radioligands. Low brain uptake is partly ascribed to efflux transporter action as well as unfavorable conformations.

Effects of sodium humate and zinc oxide used in prophylaxis of post-weaning diarrhoea on the health, oxidative stress status and fatty acid profile in weaned piglets

The aim of this study was to investigate the effects of sodium humate (HNa), applied to feed or drinking water as a partial substitution for pharmacological doses of ZnO, on clinical, biochemical and haematological indicators of health status, oxidative stress and the serum fatty acid (FA) profile in weaned piglets. Weaned piglets (32) were allocated into four groups: Control = basal diet containing 110 mg ZnO/kg; ZnO 2.5 = pharmacological dose of ZnO (2.5 g ZnO/kg); ZnO 1.7 + HNa (f) = 1.7 g ZnO and 20.0 g HNa/kg; ZnO 1.7 + HNa (w) = 1.7 g ZnO/kg and drinking water with 0.2% HNa. All ZnO treatments resulted in good performance and clinical health of piglets in contrast to Control, which contained three diarrhoeic piglets. Increased triacylglycerols in the ZnO 2.5 and ZnO 1.7 + HNa (w) groups in comparison with Control suggested increased energy metabolism after treatments. Neither total cholesterol, nor HDL and LDL were affected by treatments. Piglets treated with ZnO and HNa had (ZnO 1.7 + HNa (w)) or tended to have (ZnO 1.7 + HNa (f)) lower urea in serum. Significantly (in ZnO 2.5 and ZnO 1.7 + HNa (f)) or non-significantly (ZnO 1.7 + HNa (w)) higher haematocrit and haemoglobin levels were detected in the blood of treated piglets. Long-term ZnO 2.5 treatment significantly increased serum 8-iso-PGF<sub>2α</sub>, the most reliable biomarker of oxidative stress. Partial substitution of ZnO by HNa positively affected the oxidative status of piglets as evidenced by significant (ZnO 1.7 + HNa (w)) or non-significant (ZnO 1.7 + HNa (f)) declines in serum 8-iso-PGF<sub>2α</sub>. ZnO 2.5 treatment significantly decreased saturated (SFA), monounsaturated (MUFA) and n-3 polyunsaturated FA (PUFA) and increased PUFA n-6 and n-6/n-3 ratios in serum compared to Control. The synthesis of some physiologically significant long-chain PUFA (LC-PUFA), namely n-6 dihomo-γ-linolenic acid and n-3 eicosapentaenoic, docosapentaenoic was negatively affected by ZnO 2.5 treatment. Both ZnO 1.7 + HNa (f)/(w) treatments significantly increased the proportion of SFA and PUFA n-3 and decreased PUFA n-6 and n-6/n-3 ratios in comparison with ZnO 2.5 and the effect was more considerable in ZnO 1.7 + HNa (w). Most individual PUFA n-3 and n-6 as well as the n-6/n-3 ratio in both treatments were similar to Control. The results indicate that the partial substitution of ZnO by HNa can benefit performance and health of weaned piglets to a similar extent as a high pharmacological dose of ZnO, whereas it can decrease the oxidative stress induced by prolonged over-supplementation of ZnO. Additionally, such a treatment can eliminate the unfavourable effect of high ZnO doses on the n-6/n-3 ratio and the proportion of some physiologically significant LC-PUFA in serum. Generally, it can be concluded that the effects of feed and water HNa supplementation are similar, but are more pronounced when HNa is applied to drinking water.

Regulation of TCF/LEF promoter activity by soy phytoestrogens in 3T3‐L1 fibroblasts

The canonical Wnt pathway inhibits adipogenesis through activation of the transcription factor, TCF/LEF1. Soy phytoestogens, such as genistein (GEN) and daidzein (DAID), are paradoxically shown to both induce and repress adipogenesis. To determine if the concentration of soy phytoestrogen effects adipogenic regulation, 3T3‐L1 fibroblasts were transfected with either a estrogen receptor (ER) or TCF/LEF lentiviral reporter construct. Transfected cells were then treated with GEN or DAID at 100 pM, nM, or uM concentration. Our data showed that both ER and TCF/LEF promoter activity was 2–3 fold greater in cells treated with 100 uM of GEN or DAID (vs. vehicle). In contrast, fibroblasts treated with the pM or nM concentrations exhibited limited promoter activity. Collectively, these data indicate that the anti‐adipogenic properties of soy phytoestrogen may be attributed to high pharmacological doses.

Grape seed procyanidin extract reduces the endotoxic effects induced by lipopolysaccharide in rats

Acute inflammation is a response to injury, infection, tissue damage, or shock. Bacterial lipopolysaccharide (LPS) is an endotoxin implicated in triggering sepsis and septic shock, and LPS promotes the inflammatory response, resulting in the secretion of proinflammatory and anti-inflammatory cytokines such as the interleukins (IL-6, IL-1β, and IL-10) and tumor necrosis factor-α by the immune cells. Furthermore, nitric oxide and reactive oxygen species levels increase rapidly, which is partially due to the activation of inducible nitric oxide synthase in several tissues in response to inflammatory stimuli. Previous studies have shown that procyanidins, polyphenols present in foods such as apples, grapes, cocoa, and berries, have several beneficial properties against inflammation and oxidative stress using several in vitro and in vivo models. In this study, the anti-inflammatory and antioxidant effects of two physiological doses and two pharmaceutical doses of grape seed procyanidin extract (GSPE) were analyzed using a rat model of septic shock by the intraperitoneal injection of LPS derived from Escherichia coli. The high nutritional (75mg/kg/day) and the high pharmacological doses (200mg/kg/day) of GSPE showed anti-inflammatory effects by decreasing the proinflammatory marker NOx in the plasma, red blood cells, spleen, and liver. Moreover, the high pharmacological dose also downregulated the genes Il-6 and iNos; and the high nutritional dose decreased the glutathione ratio (GSSG/total glutathione), further illustrating the antioxidant capability of GSPE. In conclusion, several doses of GSPE can alleviate acute inflammation triggered by LPS in rats at the systemic and local levels when administered for as few as 15 days before the injection of endotoxin.

Kosttilskudd med jern ved jernmangel

A low supply of iron in the diet may result in iron deficiency and mild iron-deficiency anaemia in healthy individuals. Women are more susceptible than men because of menstrual iron loss. We compared the effect of a low dose of iron, administered as a dietary supplement, with a high pharmacological dose of iron to otherwise healthy individuals with iron deficiency and mild iron deficiency anaemia. In a randomised, double-blind trial conducted in 2000-2001, 73 women and three men with iron deficiency received either 27.6 mg of iron consisting of ferrous fumarate enriched with 13% haem iron, or 100 mg ferrosulphate daily for 12 weeks. Blood samples were analysed four times in the course of the treatment. The median ferritin value rose by 13 and 7 μg/l in the high-dose and low-dose group, respectively. The increase in ferritin was significantly higher in the high-dose than in the low dose group ( < 0.001). There was no statistically significant difference between the groups in the change in Hb, serum-iron or serum-iron binding capacity. The median haemoglobin value increased by 0.4 g/100 ml in both groups. Gastrointestinal side effects were experienced by 58% in the high-dose group and 35% in the low-dose group. Four subjects in the high-dose group and one in the low-dose group broke off the treatment because of side effects. A supplement of low-dose iron is enough to increase iron stores in cases of nutritional iron deficiency in healthy individuals and to optimise haemoglobin. High-dose iron caused the largest increase in iron stores. Low-dose iron resulted in the least side effects.

114. Neural and behavioral responses to low-grade inflammation

Neural and behavioral responses after peripheral immune challenge have been observed in numerous studies using relatively high pharmacological doses of lipopolysaccharide (LPS). However, little attention has been given to the effects of LPS dose ranges that simulate low grade-inflammation on CNS-related biology and behavior. The current studies were designed to characterize neural and behavioral responses to low-grade inflammation. Results showed burrowing and open field activities were significantly impaired following a single i.p. injection of 10 μg/kg, but not 1 μg/kg of LPS. In addition, following repeated 1 μg/kg LPS administration for 10 days, animals showed LPS-induced motor deficits in the open field starting on day 4 that continually worsened by day 10’s assessment of activity. When examining the effects of repeated low-dose LPS administration on burrowing behavior, animals showed signs of LPS tolerance following repeated doses of 1 μg/kg LPS. To correlate behavior with CNS activity, c-Fos activation was determined in the PVN, NTS, CeA, and VLM. Data revealed there was a dose-dependent activation in all brain areas examined. Furthermore, animals that received 1 μg/kg of LPS for 8 days had a PVN c-Fos activation that approached levels found following a single 10 μg/kg LPS injection. These data demonstrate neural and behavior responses can be induced by low-grade inflammation and chronic exposure to sub-threshold levels of LPS sensitizes the host to subsequent exposures of very low levels of LPS.

Rachitismes et ostéomalacies à l’âge adulte

The main cause of osteomalacia in adults is a severe and prolonged loss in vitamin D; today, it is easily recognized by 25-hydroxyvitamin D assay [25(OH)D]. The clinical context, laboratory abnormalities of the calcium-phosphate metabolism, abnormalities on medical imaging studies (radiographs, bone scan), sometimes, a very low density measured on bone densitometry can raise the possibility of mineralization disorder such as osteomalacia. Diagnosis can be confirmed by histomorphometric analysis of transiliac bone biopsy performed after double tetracyclin labeling. Cause for vitamin D deficiency should be searched and treated. Simple supplementation with vitamin D can cure these patients. Rare forms of osteomalacia, termed vitamin D-resistant rickets, are due to a genetic abnormality on an enzyme (Prader's pseudo-deficient rickets or type-1 vitamin D-resistant rickets due to the absence of alpha 1-hydroxylase) or an abnormal vitamin D receptor (VDR, type 2 vitamin D-dependent rickets) creating a resistance to the action of vitamin D in target organs. In this particular form, the treatment is difficult; it necessitates the use of high pharmacological doses of vitamin D in addition to a high calcium supplementation.

Effects of cadmium chloride and sodium selenite alone or in combination on the liver of male Sprague-Dawley rats assessed by different assays.

This study assessed the impact of either cadmium chloride (Cd) or sodium selenite (Se) alone or in combination on male Sprague-Dawley rats. For this purpose, body and liver weights, comet and TUNEL assays, histological analysis and levels of lipid peroxidation and antioxidants in liver were determined in four groups of male Sprague-Dawley rats. The rats were given subcutaneous doses of 1 mg/kg body weight (BW) of either normal saline (control=Ct) or Cd or Se or Cd plus Se (Cd+Se) on alternate days for 4 weeks. The Cd group showed increased DNA damage, apoptosis and hepatic levels of lipid peroxidation and altered histology. Conversely, the antioxidant levels in this group were decreased as compared with the control group. The Se group also showed DNA damage, apoptosis and altered histology and reduced catalase activity, but it was less severe than the Cd group. In the Cd+Se group, ameliorating effects of Se on Cd-induced changes were observed. While the Se was able to curtail the toxic effect of Cd, the Cd or Se alone were genotoxic and cytotoxic for rats receiving a high pharmacological but non-fatal dose of 1 mg/kg BW.

Role of caspase-1 and interleukin-1β in acetaminophen-induced hepatic inflammation and liver injury

Acetaminophen (APAP) overdose can result in serious liver injury and potentially death. Toxicity is dependent on metabolism of APAP to a reactive metabolite initiating a cascade of intracellular events resulting in hepatocellular necrosis. This early injury triggers a sterile inflammatory response with formation of cytokines and innate immune cell infiltration in the liver. Recently, IL-1β signaling has been implicated in the potentiation of APAP-induced liver injury. To test if IL-1β formation through caspase-1 is critical for the pathophysiology, C57Bl/6 mice were treated with the pan-caspase inhibitor Z-VD-fmk to block the inflammasome-mediated maturation of IL-1β during APAP overdose (300 mg/kg APAP). This intervention did not affect IL-1β gene transcription but prevented the increase in IL-1β plasma levels. However, APAP-induced liver injury and neutrophil infiltration were not affected. Similarly, liver injury and the hepatic neutrophilic inflammation were not attenuated in IL-1-receptor-1 deficient mice compared to wild-type animals. To evaluate the potential of IL-1β to increase injury, mice were given pharmacological doses of IL-1β after APAP overdose. Despite increased systemic activation of neutrophils and recruitment into the liver, there was no alteration in injury. We conclude that endogenous IL-1β formation after APAP overdose is insufficient to activate and recruit neutrophils into the liver or cause liver injury. Even high pharmacological doses of IL-1β, which induce hepatic neutrophil accumulation and activation, do not enhance APAP-induced liver injury. Thus, IL-1 signaling is irrelevant for APAP hepatotoxicity. The inflammatory cascade is a less important therapeutic target than intracellular signaling pathways to attenuate APAP-induced liver injury.

The expression of serum steroid sex hormones and steroidogenic enzymes following intraperitoneal administration of dehydroepiandrosterone (DHEA) in male rats

The adrenals of humans and primates could secrete large amounts of dehydroepiandrosterone (DHEA) and its sulphate ester (DHEA-S) in the circulation, which act as precursors of active steroid hormones in a long series of peripheral target intracrine tissues. The marked decline of serum DHEA and DHEA-S concentrations with age in humans has been incriminated in the development of various pathologies. Therefore, this study aims to provide detailed information on the effects of the intraperitoneal injection of DHEA on circulating steroid hormones and their metabolites and their trade-off relationship over 24 h in male rats. In this study, 100 healthy adult male Sprague–Dawley (SD) rats were randomly divided into three groups: control, 25 mg kg −1 DHEA-treated and 100 mg kg −1 DHEA-treated. The animals were sacrificed at 0, 1.5, 3, 6, 12 or 24 h, and the samples were collected for subsequent analysis. Total cholesterol (TC) markedly decreased 3 h after the administration of 100 mg kg −1 DHEA, but markedly increased 12 h after administration. The DHEA-S, progesterone (P), testosterone (T), oestradiol (E 2), cortisol (Cor) and aldosterone (Ald) concentrations also markedly increased after DHEA administration, with serum DHEA-S, T, E 2 and Cor levels peaking at 1.5 h. Over time, steroid hormone levels were depressed, but serum Cor and Ald levels were markedly elevated relative to the control group at 24 h. Furthermore, DHEA treatment produced a significant increase in P450scc, 17β-HSDIII, CYP17α and 3β-HSD mRNA expression at 1.5 h, but a decided decrease in P450scc and StAR mRNA expression at 12 and 24 h, and CYP17α and 17β-HSDIII expression at 12 h in the 100 mg kg −1 DHEA group. In total, the results of the present study indicate that DHEA at high pharmacological doses may affect steroid through an effect on steroidogenic enzymes.

The Role of Angiogenic Growth Factors in Arteriogenesis

Background/Aims: Collateral vessels restore only about 40% of the maximum dilatory reserve after femoral artery occlusion, whereas complete normalization is reached by increased fluid shear stress (FSS). We studied the role of known potent angiogenic growth factors (separately or in combination) in arteriogenesis by determining their expression in FSS-stimulated collaterals and close-to-collateral infusion of growth factor peptides in a rabbit model of femoral artery occlusion. Methods: Values of maximum collateral conductance (C_max) and post mortem angiograms were compared to those achievable by high FSS. mRNA levels of growth factor ligands and receptors were determined in FSS-stimulated collaterals. Results: Seven days after vessel occlusion, FSS-stimulated legs showed a C_max not significantly different from that of not occluded femoral arteries. Arteriogenesis was significantly less enhanced after growth factor treatment (MCP-1 86%, Ad5.1-FGF-4 75%, bFGF 72%, PDGF 64%, VEGF 50% of C_max after FSS stimulation). RT-PCR showed no differential expression of FGF receptors, but an up-regulation of VEGF-receptor-2. Conclusion: The most potent known angiogenic growth factors at high pharmacological doses reach only a fraction of the maximum conductance obtained by high FSS. Arteriogenesis differs from angiogenesis, so the main focus to markedly improve arteriogenesis should be put on the underlying mechanisms of shear stress.

Cardiac RAGE in Sepsis

See related article, pages 1239–1246 Sepsis is a systemic inflammatory response syndrome triggered by infection. The hallmarks of clinical sepsis are a broad range of systemic and organ function aberrations, including core temperature (hypothermia or hyperthermia), cardiac (rate and contractility), and respiratory and hematologic perturbations. When sepsis results in at least 1 organ failure or dysfunction, it is classified as severe sepsis. Severe sepsis with hypotension unresponsive to fluid resuscitation defines septic shock. Sepsis syndrome afflicts almost 750 000 patients in the United States each year, at a cost of almost $17 billion, and causes more than 200 000 deaths annually. The incidence of sepsis syndrome continues to rise along with the increase in life span and several other important risk factors. Sepsis without organ dysfunction is a relatively benign condition, and spontaneous recovery with conservative measures results in low in-hospital mortality (5% to 10%). Severe sepsis and septic shock carry high mortality, 30% to 50%, in spite of all that modern treatment offers.1–3 Genetic predisposing factors have been proposed including mutations in cytokine genes such as tumor necrosis factor (TNF)-α, interleukin (IL)-1, and IL-6. Most of these polymorphisms, implicated in increasing severity of sepsis, are associated with increases in these proinflammatory cytokines. Abnormalities in Toll-like receptor-4 and its signaling kinase, IRAK-4, have been associated with worse outcomes. The alarming increase in pathogens resistant to antibiotics that, in the past, were very effective in prevention and treatment of sepsis no doubt contributes to increases in severe sepsis prevalence in the elderly. The key mechanisms believed to initiate and propagate …

Activity and Regulation of Factor VIIa Analogs with Increased Potency at the Endothelial Cell Surface.

High doses of recombinant factor VIIa (FVIIa) have been found to bypass factor IX or factor VIII deficiency and ameliorate the bleeding problems associated with hemophilia patients with inhibitors. Recent studies show that FVIIa also acts as an effective hemostatic agent in other categories of patients, and thus has become a promising candidate for prevention and treatment of excessive bleeding associated with many other diseases/injuries. Although recombinant FVIIa has proven to be a very effective and safe drug in the treatment of bleeding episodes in hemophilia patients with inhibitors and other indications, a small fraction of patients may be refractory to FVIIa treatment. The reason for this is unclear at present, but it is possible that administration of very high pharmacological doses of FVIIa or use of genetically modified FVIIa molecules with increased potencies may circumvent the problem. The most dramatic effect on the activity (a 40-fold increase in proteolytic activity) of FVIIa was obtained by occupying the corresponding positions in thrombin/factor IXa for those positions 158, 296 and 298 of FVIIa (FVIIaDVQ). A FVIIa mutant in which the hydrophobic residue Met 298 was replaced with Gln (FVIIaQ) has 7-fold higher proteolytic activity. In the present study, we investigated the interactions of FVIIaQ and FVIIaDVQ with plasma inhibitors, tissue factor pathway inhibitor (TFPI) and antithrombin (AT) in solution and at the vascular endothelium. Both TFPI and AT/heparin inhibited the FVIIa variants more rapidly than the wild-type FVIIa in the absence of TF. In the presence of TF, TFPI, TFPI-Xa and AT/heparin inhibited FVIIa and FVIIa variants at similar rates. Although the wild-type FVIIa failed to generate significant amounts of factor Xa on unperturbed endothelial cells, FVIIa variants, particularly FVIIaDVQ, generated a substantial amount of factor Xa on unperturbed endothelium (1 nM of factor VIIa generated 0.3 ± 0.15 nM factor Xa/h whereas FVIIaQ and FVIIaDVQ generated 1.26 ± 0.1 nM/h and 9.48 ± 1.32 nM/h, respectively). Annexin V fully attenuated the FVIIa-mediated activation of factor X on unperturbed endothelial cells whereas anti-TF IgG had no effect. On stimulated HUVEC, FVIIa and FVIIa variants activated factor X at similar rates (30–40 nM/h). AT/heparin and TFPI-Xa inhibited the activity of FVIIa and FVIIa variants bound to endothelial cell TF in a similar fashion. AT inhibition of FVIIa bound to stimulated endothelial cells requires exogenous heparin. Interestingly, TFPI-Xa was found to inhibit the activities of both FVIIa and FVIIa analogs bound to unperturbed endothelial cells. Despite significant differences observed in factor Xa generation on native endothelium exposed to FVIIa and FVIIa analogs, no differences were found in thrombin generation when cells were exposed to FVIIa or FVIIa analogs under plasma mimicking conditions, probably due to limited availability of anionic phospholipids and/or putative factor Xa and Va binding sites on their cell surface. Over all, our present data suggest that although FVIIa variants may generate factor Xa on native endothelium, the resultant factor Xa does not lead to enhanced thrombin generation on native endothelium compared to FVIIa. These data should reduce potential concerns about whether the use of FVIIa variants triggers unwanted coagulation on native endothelium, and may facilitate the development of FVIIa analogs as effective therapeutic agents in near future for treatment of patients with bleeding disorders.

Oestrogen Synthesis in the Hippocampus: Role in Axon Outgrowth

Ovarian oestrogens have been postulated to be neuroprotective. It has also been shown that considerable amounts of oestrogens are synthesised in hippocampal neurones. In the present study, we focused on a potential role of hippocampus-derived oestradiol compared to gonad-derived oestradiol on axon outgrowth of hippocampal neurones. To address the role of hippocampus-derived oestradiol, we inhibited oestrogen synthesis by treatment of neonatal hippocampal cell cultures with letrozole, a specific aromatase inhibitor. As an alternative, we used siRNA against steroidogenic acute regulatory protein (StAR). Axon outgrowth and GAP-43 expression were significantly down-regulated in response to letrozole and in siRNA-StAR transfected cells. The effects after inhibition of oestrogen synthesis in response to letrozole and in siRNA-StAR transfected cells were reversed by oestrogen supplementation. No difference was found between ovariectomised animals, cycling animals at pro-oestrus and ovariectomised and subsequently oestradiol-treated animals. However, high pharmacological doses of oestradiol promoted axon outgrowth, which was possible to abolish by the oestrogen receptor antagonist ICI 182,780. Our results show that oestradiol-induced neurite outgrowth is very likely mediated by genomic oestrogen receptors and requires higher doses of oestradiol than physiological serum concentrations derived from the gonads.

The Effects of Zinc on the Olfactory Neuroepithelium and Olfactory Bulbs of the Sprague-Dawley Rat after oral Administration of Zinc-Gluconate Trihydrate

The most frequent causes of upper respiratory infections are human rhinoviruses. The nasopharyngeal area, which includes the respiratory epithelium, mucosa, and the olfactory neuroepithelium (ONe), is a first-line of defense against airborne viruses and allergens, some of which manage to penetrate the nasal mucosa and invade the tissues of the nasal respiratory epithelium. Biochemical evidence from several studies suggests that zinc is an effective cold treatment and that over-the-counter (OTC) zinc-gluconate compounds may provide the high pharmacologic doses of zinc needed to act as an effective means of treating and reducing the duration and severity of symptoms of the common cold. A series of male Sprague-Dawley rats were fed an oral preparation of zinc-gluconate trihydrate or received the equivalent through drinking water to investigate the potential cytotoxic and/or neurotoxic insult to the olfactory receptor cells and other tissue in the ONe and afferent neuronal pathways. Coronal sections of the rat ONe and corresponding olfactory bulbs showed consistent cellular and tissue damage of increasing severity that correlated with the duration of treatment with the zinc compound when compared with the control group animals. The results of this analysis indicate that the repeated oral administration of such zinc-containing compounds have neurotoxic effects on the ONe and to the mitral cells in the olfactory bulbs of treated rats. These findings point toward the need for increased investigation into the potential deleterious effects of zinc-containing compounds to humans as well.