Erythropoietin mimics the acute phase response in critical illness

Background: In a prospective observational study, we examined the temporal relationships between serum erythropoietin (EPO) levels, haemoglobin concentration and the inflammatory response in critically ill patients with and without acute renal failure (ARF). Patients and method Twenty-five critically ill patients, from general and cardiac intensive care units (ICUs) in a university hospital, were studied. Eight had ARF and 17 had normal or mildly impaired renal function. The comparator group included 82 nonhospitalized patients with normal renal function and varying haemoglobin concentrations. In the patients, levels of haemoglobin, serum EPO, C-reactive protein, IL-1β, IL-6, serum iron, ferritin, vitamin B12 and folate were measured, and Coombs test was performed from ICU admission until discharge or death. Concurrent EPO and haemoglobin levels were measured in the comparator group. Results: EPO levels were initially high in patients with ARF, falling to normal or low levels by day 3. Thereafter, almost all ICU patients demonstrated normal or low EPO levels despite progressive anaemia. IL-6 exhibited a similar initial pattern, but levels remained elevated during the chronic phase of critical illness. IL-1β was undetectable. Critically ill patients could not be distinguished from nonhospitalized anaemic patients on the basis of EPO levels. Conclusion: EPO levels are markedly elevated in the initial phase of critical illness with ARF. In the chronic phase of critical illness, EPO levels are the same for patients with and those without ARF, and cannot be distinguished from noncritically ill patients with varying haemoglobin concentrations. Exogenous EPO therapy is unlikely to be effective in the first few days of critical illness.

Synthesis of unsaturated aminopyranosides as possible transition state mimics for glycosidases

Four unsaturated aminopyranosides have been prepared as possible transition-state mimics targeted towards carbohydrate processing enzymes. The conformations of the protonated aminosugars have been investigated by molecular modelling and their ability to inhibit alpha- and beta-glucosidases and an a-mannosidase have been probed. Two targets proved moderate inhibitors of alpha-glucosidases from Brewer's yeast and Bacillus stearothennophilus.

Anti-platelet effect of aspirin is substantially reduced after administration of heparin during carotid endarterectomy

OBJECTIVES: Aspirin therapy is usually continued throughout the perioperative period to reduce the risk for thromboembolic stroke and myocardial infarction after carotid endarterectomy (CEA). Aspirin irreversibly binds cyclooxygenase-1, thereby reducing platelet aggregation for the lifetime of each platelet. However, recent research from this unit has shown that aggregation in response to arachidonic acid increases significantly, but transiently, during CEA, which suggests that the anti-platelet effect of aspirin is temporarily reversed. The purpose of the current study was to determine when this phenomenon occurs and to identify the possible mechanisms involved. METHODS: Platelet aggregation was measured in platelet-rich plasma from 41 patients undergoing CEA who were stabilized with 150 mg of aspirin daily. Blood was taken at 8 time points: before anesthesia, after anesthesia, before heparinization, 3 minutes after heparinization, 3 minutes after shunt insertion, 10 minutes after flow restoration, 4 hours postoperatively, and 24 hours postoperatively. Platelet aggregation was also measured at similar times in a group of 18 patients undergoing peripheral angioplasty without general anesthesia. RESULTS: All patient platelets were effectively inhibited by aspirin at the start of the operation. There was a significant intraoperative increase in platelet response to arachidonic acid in both groups of patients, which occurred within 3 minutes of administration of unfractionated heparin. In the CEA group this resulted in a greater than 10-fold increase in mean aggregation, to 5 mmol/L of arachidonic acid (5 mmol/L), rising from 3.9% +/- 2.2% preoperatively to 45.1% +/- 29.3% after administration of heparin ( P <.0001). This increased aggregation persisted into the early postoperative period, but by 24 hours post operation aggregation had returned to near preoperative values. Aggregation in response to other platelet agonists (adenosine diphosphate, thrombin receptor agonist peptide) showed only a small increase at the same time, which could be accounted for by a parallel increase in the level of spontaneous aggregation. CONCLUSION: Administration of heparin significantly increases platelet aggregation in response to arachidonic acid, despite adequate inhibition by aspirin administered preoperatively. This apparent reversal in anti-platelet activity persisted into the immediate early postoperative period, and could explain why a small proportion of patients are at increased risk for acute cardiovascular events after major vascular surgery, despite aspirin therapy.

Synthesis of S-linked carbohydrate analogues via a Ferrier reaction

In this work, the synthetic utility of the Ferrier reaction to access S-linked disaccharides and S-linked glycoamino acids has been probed. Significantly, entry to a range of 1,4- and 1,6-S-linked disaccharides has been achieved using glycals derived from glucose and galactose, and sulfur containing coupling partners derived from methyl α-d-glucopyranoside. Access to S-linked glycoamino acids and glycopeptides has also been achieved using protected cysteine and homocysteine coupling partners within the Ferrier reaction. Functionalisation of the Ferrier products, for example, via dihydroxylation using OsO4 or amino acid coupling, and deprotection of the targets have also been achieved. In this way, entry to materials of interest as mimics of biologically interesting disaccharides and glycopeptides has been realised, including targets derived from rare sugars such as talopyranose and gulopyranose.

Bilateral high-frequency stimulation of the subthalamic nucleus on attentional performance: transient deleterious effects and enhanced motivation in both intact and parkinsonian rats.

It is now well established that subthalamic nucleus high-frequency stimulation (STN HFS) alleviates motor problems in Parkinson's disease. However, its efficacy for cognitive function remains a matter of debate. The aim of this study was to assess the effects of STN HFS in rats performing a visual attentional task. Bilateral STN HFS was applied in intact and in bilaterally dopamine (DA)-depleted rats. In all animals, STN HFS had a transient debilitating effect on all the variables measured in the task. In DA-depleted rats, STN HFS did not alleviate the deficits induced by the DA lesion such as omissions and latency to make correct responses, but induced perseverative approaches to the food magazine, an indicator of enhanced motivation. In sham-operated controls, STN HFS significantly reduced accuracy and induced perseverative behaviour, mimicking partially the effects of bilateral STN lesions in the same task. These results are in line with the hypothesis that STN HFS only partially mimics inactivation of STN produced by lesioning and confirm the motivational exacerbation induced by STN inactivation.

Quality of medicines stored together in multi-compartment compliance aids

Background and Objective: Dispensing medicines into compliance aids is a common practice in pharmacy contrary to manufacturers’ advice and studies have shown the appearance of light-sensitive tablets is compromised by such storage; we previously found evidence of reduced bioavailability at elevated temperature and humidity. Our objective was to examine the physicochemical stability of two generic atenolol tablets in different compliance aids and with aspirin co-storage at room temperature and at 40 °C/75% relative humidity. Methods: The physicochemical stability of atenolol tablets was evaluated after 28 days of storage and compared with controls by examining visual appearance, weight, disintegration, dissolution, friability and hardness to accepted standards and using a previously validated HPLC method for chemical assay. Results and Discussion: The response to storage was brand-dependent and not straightforward. With one make of atenolol (Alpharma), storage in compliance aids even at room temperature impacted on physical stability, reducing tablet hardness, with storage in Dosett® exerting a greater impact than storage in Medidos® (t-test P < 0·001). Co-storage at elevated temperature and humidity also impacted on the appearance of non-coated aspirin tablets (Angette™). The chemical stability of atenolol was not affected and we did not find evidence of changes to bioavailability with either make. Certainly data for one atenolol make (CP Pharmaceuticals) co-stored with aspirin (Angette™ and Nu-Seals) in both compliance aids at room temperature provided evidence of short-term stability. But medicines are dispensed into compliance aids in multi-factorial ways so our study highlights not only the lack of evidence but also a realization that evidence to support real practice may not be accomplished through research. Conclusion: Reassuring practitioners of the continued stability of medicines in compliance aids under the countless condition in which they are dispensed in practice may requires a different approach involving medical device regulators and more definitive professional guidance.

In vitro cumulative gas production techniques: History, methodological considerations and challenges

Methodology used to measure in vitro gas production is reviewed to determine impacts of sources of variation on resultant gas production profiles (GPP). Current methods include measurement of gas production at constant pressure (e.g., use of gas tight syringes), a system that is inexpensive, but may be less sensitive than others thereby affecting its suitability in some situations. Automated systems that measure gas production at constant volume allow pressure to accumulate in the bottle, which is recorded at different times to produce a GPP, and may result in sufficiently high pressure that solubility of evolved gases in the medium is affected, thereby resulting in a recorded volume of gas that is lower than that predicted from stoichiometric calculations. Several other methods measure gas production at constant pressure and volume with either pressure transducers or sensors, and these may be manual, semi-automated or fully automated in operation. In these systems, gas is released as pressure increases, and vented gas is recorded. Agitating the medium does not consistently produce more gas with automated systems, and little or no effect of agitation was observed with manual systems. The apparatus affects GPP, but mathematical manipulation may enable effects of apparatus to be removed. The amount of substrate affects the volume of gas produced, but not rate of gas production, provided there is sufficient buffering capacity in the medium. Systems that use a very small amount of substrate are prone to experimental error in sample weighing. Effect of sample preparation on GPP has been found to be important, but further research is required to determine the optimum preparation that mimics animal chewing. Inoculum is the single largest source of variation in measuring GPP, as rumen fluid is variable and sampling schedules, diets fed to donor animals and ratios of rumen fluid/medium must be selected such that microbial activity is sufficiently high that it does not affect rate and extent of fermentation. Species of donor animal may also cause differences in GPP. End point measures can be mathematically manipulated to account for species differences, but rates of fermentation are not related. Other sources of inocula that have been used include caecal fluid (primarily for investigating hindgut fermentation in monogastrics), effluent from simulated rumen fermentation (e.g., 'Rusitec', which was as variable as rumen fluid), faeces, and frozen or freeze-dried rumen fluid (which were both less active than fresh rumen fluid). Use of mixtures of cell-free enzymes, or pure cultures of bacteria, may be a way of increasing GPP reproducibility, while reducing reliance on surgically modified animals. However, more research is required to develop these inocula. A number of media have been developed which buffer the incubation and provide relevant micro-nutrients to the microorganisms. To date, little research has been completed on relationships between the composition of the medium and measured GPP. However, comparing GPP from media either rich in N or N-free, allows assessment of contributions of N containing compounds in the sample. (c) 2005 Published by Elsevier B.V.

Low molecular weight heparin significantly reduces embolisation after carotid endarterectomy - a randomised controller trial

Objectives The administration of unfractionated heparin (UFH) prior to carotid clamping during carotid endarterectomy (CEA) transiently increases the platelet aggregation response to arachidonic acid (AA) despite the use of aspirin. We hypothesized that this phenomenon might be reduced by using low molecular weight heparin (LMWH) resulting in fewer emboli in the early post-operative period. Methods 183 aspirinated patients undergoing CEA were randomised to 5000 IU UFH (n = 91) or 2500 IU LMWH (dalteparin, n = 92) prior to carotid clamping. End-points were: transcranial Doppler (TCD) measurement of embolisation, effect on bleeding and platelet aggregation to AA and adenosine 5′-diphosphate (ADP). Results Patients randomised to UFH had twice the odds of experiencing a higher number of emboli in the first 3 h after CEA, than those randomised to LMWH (p = 0.04). This was not associated with increased bleeding (mean time from flow restoration to operation end: 23 min (UFH) vs. 24 min (LMWH), p = 0.18). Platelet aggregation to AA increased significantly following heparinisation, but was unaffected by heparin type (p = 0.90). The platelets of patients randomised to LMWH exhibited significantly lower aggregation to ADP compared to UFH (p < 0.0001). Conclusions Intravenous LMWH is associated with a significant reduction in post-operative embolisation without increased bleeding. The higher rate of embolisation seen with UFH may be mediated by increased platelet aggregation to ADP, rather than to AA.

The neurokinin 1 receptor: a potential new target for anti-platelet therapy?

The important role of platelets in the development of arterial thrombosis and cardiovascular disease is well established. Current treatments for arterial thrombosis include anti-platelet agents such as aspirin, thienopyridines and glycoprotein IIb-IIIa inhibitors. Despite these drugs being effective there remains a substantial unmet clinical demand for more effective therapeutic approaches, which may reflect the existence of alternative underlying regulatory mechanisms to those already targeted. Recent publications have demonstrated a key role for tachykinins in the positive feedback regulation of platelet aggregation and thrombus formation. The pro-thrombotic effects of tachykinins on platelets are mediated through the neurokinin 1 receptor, which may therefore offer a novel therapeutic drug target in the prevention and the treatment of arterial thrombosis.

Flavonoids inhibit the platelet TxA(2) signalling pathway and antagonize TxA(2) receptors (TP) in platelets and smooth muscle cells

What is already known about this subject center dot Flavonoids are largely recognized as potential inhibitors of platelet function, through nonspecific mechanisms such as antioxidant activity and/or inhibition of several enzymes and signalling proteins. center dot In addition, we, and few others, have shown that certain antiaggregant flavonoids may behave as specific TXA2 receptor (TP) ligands in platelets. center dot Whether flavonoids interact with TP isoforms in other cell types is not known, and direct evidence that flavonoid-TP interaction inhibits signalling downstream TP has not been shown. What this study adds center dot This study first demonstrates that certain flavonoids behave as ligands for both TP isoforms, not only in platelets, but also in human myometrium and in TP-transfected HEK 293T cells. center dot Differences in the effect of certain flavonoids in platelet signalling, induced by either U46619 or thrombin, suggest that abrogation of downstream TP signalling is related to their specific blockage of the TP, rather than to a nonspecific effect on tyrosine kinases or other signalling proteins. Flavonoids may affect platelet function by several mechanisms, including antagonism of TxA(2) receptors (TP). These TP are present in many tissues and modulate different signalling cascades. We explored whether flavonoids affect platelet TP signalling, and if they bind to TP expressed in other cell types. Platelets were treated with flavonoids, or other selected inhibitors, and then stimulated with U46619. Similar assays were performed in aspirinized platelets activated with thrombin. Effects on calcium release were analysed by fluorometry and changes in whole protein tyrosine phosphorylation and activation of ERK 1/2 by Western blot analysis. The binding of flavonoids to TP in platelets, human myometrium and TP alpha- and TP beta-transfected HEK 293T cells was explored using binding assays and the TP antagonist H-3-SQ29548. Apigenin, genistein, luteolin and quercetin impaired U46619-induced calcium mobilization in a concentration-dependent manner (IC50 10-30 mu M). These flavonoids caused a significant impairment of U46619-induced platelet tyrosine phosphorylation and of ERK 1/2 activation. By contrast, in aspirin-treated platelets all these flavonoids, except quercetin, displayed minor effects on thrombin-induced calcium mobilization, ERK 1/2 and total tyrosine phosphorylation. Finally, apigenin, genistein and luteolin inhibited by > 50% H-3-SQ29548 binding to different cell types. These data further suggest that flavonoids may inhibit platelet function by binding to TP and by subsequent abrogation of downstream signalling. Binding of these compounds to TP occurs in human myometrium and in TP-transfected HEK 293T cells and suggests that antagonism of TP might mediate the effects of flavonoids in different tissues.

A system for studying epithelial-stromal interactions reveals distinct inductive abilities of stromal cells from benign prostatic hyperplasia and prostate cancer

The development of normal and abnormal glandular structures in the prostate is controlled at the endocrine and paracrine levels by reciprocal interactions between epithelium and stroma. To study these processes it is useful to have an efficient method of tissue acquisition for reproducible isolation of cells from defined histologies. Here we assessed the utility of a standardized system for acquisition and growth of prostatic cells from different regions of the prostate with different pathologies, and we compared the abilities of stromal cells from normal peripheral zone (PZ-S), benign prostatic hyperplasia (BPH-S), and cancer (CA-S) to induce the growth of a human prostatic epithelial cell line (BPH-1) in vivo. Using the tissue recombination method, we showed that grafting stromal cells (from any histology) alone, or BPH-1 epithelial cells alone produced no visible grafts. Recombining PZ-S with BPH-1 cells also produced no visible grafts (n = 15). Recombining BPH-S with BPH-1 cells generated small, well-organized and sharply demarcated grafts approximately 3-4 mm in diameter (n = 9), demonstrating a moderate inductive ability of BPH-S. Recombining CA-S with BPH-1 cells generated highly disorganized grafts that completely surrounded the host kidney and invaded into adjacent renal tissue, demonstrating induction of an aggressive phenotype. We conclude that acquisition of tissue from toluidine blue dye stained specimens is an efficient method to generate high quality epithelial and/or stromal cultures. Stromal cells derived by this method from areas of BPH and cancer induce epithelial cell growth in vivo which mimics the natural history of these diseases.

Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit vascular smooth muscle cell proliferation via differential effects on the cell cycle

Abnormal vascular smooth muscle cell (VSMC) proliferation plays an important role in the pathogenesis of both atherosclerosis and restenosis. Recent studies suggest that high-dose salicylates, in addition to inhibiting cyclooxygenase activity, exert an antiproliferative effect on VSMC growth both in-vitro and in-vivo. However, whether all non-steroidal anti-inflammatory drugs (NSAIDs) exert similar anti proliferative effects on VSMCs, and do so via a common mechanism of action, remains to be shown. In this study, we demonstrate that the NSAIDs aspirin, sodium salicylate, diclofenac, ibuprofen, indometacin and sulindac induce a dose-dependent inhibition of proliferation in rat A10 VSMCs in the absence of significant cytotoxicity. Flow cytometric analyses showed that exposure of A10 cells to diclofenac, indometacin, ibuprofen and sulindac, in the presence of the mitotic inhibitor, nocodazole, led to a significant G0/G1 arrest. In contrast, the salicylates failed to induce a significant G1 arrest since flow cytometry profiles were not significantly different from control cells. Cyclin A levels were elevated, and hyperphosphorylated p107 was present at significant levels, in salicylate-treated A10 cells, consistent with a post-G1/S block, whereas cyclin A levels were low, and hypophosphorylated p107 was the dominant form, in cells treated with other NSAIDs consistent with a G1 arrest. The ubiquitously expressed cyclin-dependent kinase (CDK) inhibitors, p21 and p27, were increased in all NSAID-treated cells. Our results suggest that diclofenac, indometacin, ibuprofen and sulindac inhibit VSMC proliferation by arresting the cell cycle in the G1 phase, whereas the growth inhibitory effect of salicylates probably affects the late S and/or G2/M phases. Irrespective of mechanism, our results suggest that NSAIDs might be of benefit in the treatment of certain vasculoproliferative disorders.

Inhibition of vascular smooth muscle cell proliferation by non-steroidal anti-inflammatory drugs

Abnormal vascular smooth muscle cell (VSMC) proliferation is known to play an important role in the pathogenesis of atherosclerosis, restenosis and instent stenosis. Recent studies suggest that salicylates, in addition to inhibiting cyclooxygenase activity, exert an antiproliferative effect on VSMC growth both in vitro and in vivo. However, whether all non-steroidal anti-inflammatory drugs (NSAID) exert similar antiproliferative effects on VSMCs, and do so via a common mechanism of action, remains unknown. In the present study, we demonstrated that the NSAIDs, aspirin, ibuprofen and sulindac induced a dose-dependent inhibition of proliferation in rat A10 VSMCs (IC50 = 1666 mumol/L, 937 mumol/L and 520 mumol/L, respectively). These drugs did not show significant cytotoxic effects as determined by LDH release assay, even at the highest concentrations tested (aspirin, 5000 mumol/L; ibuprofen, 2500 mumol/L; and sulindac, 1000 mumol/L). Flow cytometric analyses showed that a 48 h exposure of A10 VSMCs to ibuprofen (1000 mumol/L) and sulindac (750 mumol/L) led to a significant G1 arrest (from 68.7 +/- 2.0% of cells in G1 to 76.6 +/- 2.2% and 75.8 +/- 2.2%, respectively, p < 0.05). In contrast, aspirin (2500 mumol/L) failed to induce a significant G1 arrest (68.1 +/- 5.2%). Clearer evidence of a G1 block was obtained by treatment of cells with the mitotic inhibitor, nocodazole (40 ng/ml), for the final 24 h of the experiment. Under these conditions, aspirin still failed to induce a G1 arrest (from 25.9 +/- 10.9% of cells in G1 to 19.6 +/- 2.3%) whereas ibuprofen and sulindac led to a significant accumulation of cells in G1(51.8% +/- 17.2% and 54.1% +/- 10.6%, respectively, p < 0.05). These results indicate that ibuprofen and sulindac inhibit VSMC proliferation by arresting the cell cycle in the G1 phase whereas the effect of aspirin appears to be independent of any special phase of the cell cycle. Irrespective of mechanism, our results suggest that NSAIDs might be of benefit to the treatment of vascular proliferative disorders.

Sialyloligosaccharides inhibit cholera toxin binding to the GM1 receptor

It is recognised that cholera toxin (Ctx) is a significant cause of gastrointestinal disease globally, particularly in developing countries where access to uncontaminated drinking water is at a premium. Ctx vaccines are prohibitively expensive and only give short-term protection. Consequently, there is scope for the development of alternative control strategies or prophylactics. This may include the use of oligosaccharides as functional mimics for the cell-surface toxin receptor (GM I). Furthermore, the sialic acid component of epithelial receptors has already been shown to contribute significantly to the adhesion and pathogenesis of Ctx. Here, we demonstrate the total inhibition of Ctx using GM1-competitive ELISA with 25 mg mL(-1) of a commercial preparation of sialyloligosaccharides (SOS). The IC50 value was calculated as 5.21 mg mL(-1). One-hundred percent inhibition was also observed at all concentrations of Ctx-HRP tested with 500 ng mL(-1) GM1-OS. Whilst SOS has much lower affinity for Ctx than GM1-OS, the commercial preparation is impure containing only 33.6% carbohydrate; however, the biantennary nature of SOS appears to give a significant increase in potency over constituent monosaccahride residues. It is proposed that SOS could be used as a conventional food additive, such as in emulsifiers, stabilisers or sweeteners, and are classified as nondigestible oligosaccharides that pass into the small intestine, which is the site of Ctx pathogenesis. (C) 2008 Elsevier Ltd. All rights reserved.

Galactooligosaccharides (GOS) inhibit Vibrio cholerae toxin binding to its GM1 receptor

It is widely reported that cholera toxin (Ctx) remains a significant cause of gastrointestinal disease globally, particularly in developing countries where access to clean drinking water is at a premium. Vaccines are prohibitively expensive and have shown only short-term protection. Consequently, there is scope for continued development of novel treatment strategies. One example is the use of galactooligosaccharides (GOS) as functional mimics for the cell-surface toxin receptor (GM1). In this study, GOS fractions were fractionated using cation exchange chromatography followed by structural characterization using a combination of hydrophilic interaction liquid chromatography (HILIC) and electrospray ionization mass spectrometry (ESI-MS) such that their molecular weight profiles were known. Each profile was correlated against biological activity measured using a competitive inhibitory GM1-linked ELISA. GOS fractions containing > 5% hexasaccharides (DP6) exhibited > 90% binding, with EC50 values between 29.27 and 56.04 mg/mL. Inhibition by GOS DP6, was dose dependent, with an EC50 value of 5.10 mg/mL (5.15 mu M MW of 990 Da). In removing low molecular weight carbohydrates that do possess prebiotic, nutraceutical, and/or biological properties and concentrating GOS DP5 and/or DP6, Ctx antiadhesive activity per unit of (dry) weight was improved. This could be advantageous in the manufacture of pharmaceutical or nutraceutical formulations for the treatment or prevention of an acute or chronic disease associated with or caused by the adhesion and/or uptake of a Ctx or HLT.