Determination of second messengers and protein kinase C in bone marrow derived macrophages stimulated with a bacterial lipopeptide

The synthetic lipopeptide Pam3Cys-Ala-Gly, an analogue of the N-terminal part of bacterial lipoprotein, constitutes a potent macrophage activator. The role of protein kinase C (PKC) in lipopeptide induced signal transduction was investigated. As determined by enzymatic and immunochemical methods, translocation of PKC could not be observed in lipopeptide stimulated bone marrow derived macrophages. Our studies showed that the membrane-associated form of PKC displayed different characteristics than the cytosolic form. The second messengers, inositoltrisphosphate, cAMP and cGMP, did not seem to be involved in signal transduction. Unlike LPS, Pam3Cys-Ala-Gly induced a rapid rise in cytosolic Ca2+, which was due to an influx of extracellular calcium as well as to a redistribution of intracellular calcium. The data suggest that one major intracellular signal transduction mechanism initiated by lipopeptide consists of altering internal Ca2+ concns.     

Costs and efficacy of three different esomeprazole treatment strategies for long-term management of gastro-oesophageal reflux symptoms in primary care

BACKGROUND: A prospective, open, randomized multi-centre study with parallel group design was conducted in 155 general practice clinics, and included 1357 endoscopically uninvestigated patients with symptoms suggestive of gastro-oesophageal reflux disease. AIM: To assess the differences in direct medical costs between a patient-controlled on-demand treatment strategy with esomeprazole, 20 mg daily, and general practitioner-controlled intermittent treatment strategies with esomeprazole, 40 mg daily, for either 2 or 4 weeks. Secondary objectives were to measure other costs, total costs, patient satisfaction and time to first relapse. METHODS: The primary cost analysis was carried out as a cost minimization analysis, comparing the direct medical costs in patients allocated to on-demand treatment vs. those in patients allocated to either of the intermittent treatment strategies. RESULTS: The mean direct medical costs were 182, 221 and 195 euros for patient-controlled on-demand treatment and 2 weeks and 4 weeks of general practitioner-controlled intermittent treatment, respectively, showing no statistically significant difference. The comparable mean total costs were 211, 344 and 300 euros, i.e. significantly lower for patients treated on-demand compared with either of the general practitioner-controlled intermittent treatment strategies. CONCLUSIONS: The mean total costs, but not the mean direct medical costs, were higher in general practitioner-controlled intermittent treatment strategies with esomeprazole compared with a patient-controlled on-demand treatment strategy.     

Comparative studies between rates of incorporation of branched-chain amino acids and their alpha-ketoanalogues into rat tissue proteins under different dietary conditions

Male albino rats (110-120 g) were fed for 10 days on an amino acid diet low in nitrogen (nitrogen = 1.05%) devoid of valine, leucine and isoleucine and supplemented with branched-chain alpha-ketoacids (9.4%) (BCKA-diet). Pair-fed controls received an isocaloric diet (AA-diet) which contained the three branched-chain amino acids (1.4%) instead of the alpha-ketoacids (nitrogen = 1.2%). A third group was fed on a standard diet. Measuring rates of incorporation of radioactive leucine, valine and their corresponding alpha-ketoacids into liver, kidney, heart and brain proteins of rats fed on a standard diet revealed that in liver, branched-chain alpha-ketoacids are incorporated to a lesser extent than the corresponding amino acids. The same was also observed with the BCKA-diet, while the AA-diet reduced BCAA incorporation with the consecutive improvement of the relative incorporation of BCKA over that of BCAA. Injection of branched-chain amino- and alpha-ketoacids results in equal rates of incorporation in kidney and heart proteins. Injecting branched-chain alpha-ketoacids leads to higher incorporation rates in brain than injecting branched-chain amino acids. Thus rates of incorporation of branched-chain alpha-ketoacids differ dependent on the tissue and on the diet applied. They are not consistent with those of branched-chain amino acids.     

Effect of benzoate on glycine metabolism in rats fed branched-chain alpha-ketoacids

Male albino rats (110-120 g) were fed for 9 days an amino acid diet low in nitrogen (nitrogen, 1.05%) devoid of valine, leucine and isoleucine and supplemented with branched-chain alpha-ketoacids (9.4%) (BCKA diet). Pair-fed controls received an isocaloric diet (AA diet), which contained the three branched-chain amino acids (1.4%) instead of the alpha-ketoacids (nitrogen, 1.2%). A third group was fed a standard diet. The animals were divided into three groups of 12 animals, and 6 animals of each group were injected with benzoate (210 mumol/100 g) for 9 days. On day 10 of the experiment the activity of the glycine cleavage system and glycine concentrations were measured in liver; glycine, serine, ammonia, urea, glutamine and glutamate concentrations were determined in plasma on day 10 and determinations of urinary urea, ammonia, glutamine, glutamate, hippuric acid and benzoate were carried out on days 0 and 10 of the experiment. Feeding BCKA led to an increase of plasma and hepatic glycine levels. Activity of hepatic glycine synthesis was reduced in the BCKA-fed group, whereas compared to the AA-fed group no increase in the decarboxylation activity was observed. Injection of benzoate led to an increase in hepatic glycine decarboxylation and glycine synthesis activity in the BCKA-fed group. Plasma glycine levels did not fall, and there was no change in any of the metabolites measured in urine and plasma.     

Studies on the effect of xylitol on oxalate formation.

The excretion of [¹⁴C]oxalate after parenteral administration of [U-¹⁴C]xylitol, [U-¹⁴]fructose, [U-¹⁴C]glucose [U-¹⁴C]sorbitol and [U-¹⁴C]glycine has been studied in normally fed rats. All of these compounds were about equally effective as precursors of the urinary oxalate. The effect of xylitol on the NAD⁺-dependent catalytic oxidation of glyoxylate to oxalate has also been investigated in rat liver cytosol, this reaction being the last step on the oxalate biosynthetic pathway. Xylitol slightly decreased oxalate production from glyoxylate in this system. These results are discussed in relation to the observation that the clinical use of xylitol for parenteral nutrition is sometimes complicated by renal failure with deposits of calcium oxalate in the renal tubules.     

Regulation of adenosine receptor subtypes during cultivation of human monocytes: role of receptors in preventing lipopolysaccharide-triggered respiratory burst

Adenosine is a potent anti-inflammatory agent that modulates the function of cells involved in the inflammatory response. Here we show that it inhibits lipopolysaccharide (LPS)-induced formation of reactive oxygen intermediates (ROI) in both freshly isolated and cultured human monocytes. Blocking of adenosine uptake and inactivation of the adenosine-degrading enzyme adenosine deaminase enhanced the inhibitory action of adenosine, indicating that both pathways regulate the extracellular adenosine concentration. Adenosine-mediated inhibition could be reversed by XAC (xanthine amine congener), an antagonist of the adenosine receptor A(2A), and MRS 1220 [N-9-chloro-2-(2-furanyl)[1, 2, 4]-triazolo[1,5-c]quinazolin-5-benzeneacetamide], an A(3) receptor antagonist, in both cell populations, while DPCPX (1,3-dipropyl-8-cyclopentylxanthine), an A(1) receptor antagonist, had no effect. Similar to what was seen with adenosine, CGS 21680, an A(2A) and A(3) receptor agonist, and IB-MECA, a nonselective A(1) and A(3) receptor agonist, dose dependently prevented ROI formation, indicating the involvement of A(3) and probably also A(2A) in the suppressive effect of adenosine. Pretreatment of monocytes with adenosine did not lead to changes in the LPS-induced increase in intracellular calcium levels ([Ca(2+)](i)). Thus, participation of [Ca(2+)](i) in the action of adenosine seems unlikely. The adenosine-mediated suppression of ROI production was found to be more pronounced when monocytes were cultured for 18 h, a time point at which changes in the mRNA expression of adenosine receptors were observed. Most prominent was the increase in the A(2A) receptor mRNA. These data demonstrate that cultivation of monocytes is accompanied by changes in the inhibitory action of adenosine mediated by A(3) and probably also the A(2A) receptor and that regulation of adenosine receptors is an integral part of the monocyte differentiation program.     

Pentoxyphylline and propentophylline are inhibitors of TNF-α release in monocytes activated by advanced glycation endproducts

Summary. Non-enzymatic glycation of proteins with reducing sugars and subsequent transition metal-catalyzed oxidation leads to the formation of protein-bound advanced glycation endproducts (AGEs). They accumulate on long-lived protein deposits inducing senile plaques in Alzheimers disease. AGE-modified proteins are able to activate microglia and astroglia and can cause chronic inflammation. The aim of the present study was to confirm the stimulatory effect of different AGEs on TNF- release in human monocytes. Furthermore, the effects of four xanthine derivatives on AGE-induced TNF- release were investigated. We show that chicken egg albumin-AGEs prepared with glucose and chicken egg albumin-AGEs prepared with methylglyoxal dose-dependently induce TNF- release. The xanthine derivatives pentoxyphylline and propentophylline attenuate AGE-induced TNF- release in a dose-dependent manner. Theophylline at low concentrations slightly stimulated TNF- release whereas caffeine had no effect. The inhibition of the AGE-induced TNF- release by pentoxyphylline and propentophylline provides interesting pharmacological strategies for diseases with local neuroinflammation such as Alzheimers disease.     

Predictors of patient response to pulmonary thromboendarterectomy

OBJECTIVE: We sought to identify imaging features that help predict surgical success in patients undergoing thromboendarterectomy. MATERIALS AND METHODS: Thirty-nine consecutive patients who underwent pulmonary angiography and thromboendarterectomy during 1995 and 1996 were included. Thirty-four underwent helical CT angiography. Measurements of postoperative pulmonary vascular resistance were compared with preoperative imaging features and preoperative pulmonary vascular resistance. RESULTS: The best imaging indicators of a relatively high postoperative pulmonary vascular resistance were the extent of small vessel disease identified on CT angiograms as segments with abnormal perfusion but normal segmental arteries (p = 0.005) and the extent of central disease (p = 0.015). Combined with preoperative pulmonary vascular resistance, these features had a strong correlation with postoperative outcome (p = 0.0005). Segmental arterial disease seen on both conventional angiography and CT angiography correlated poorly with surgical outcome. CONCLUSION: In patients with chronic thromboembolic pulmonary hypertension, CT angiographic evidence of extensive central vessel disease and limited small vessel involvement indicates a favorable surgical outcome.     

Michael adducts of palmitoylascorbic acid: effects on the oxidative burst of neutrophils and the production of tumor necrosis factor-alpha in monocytes

The effects of Michael adducts of 6-O-palmitoyl-L-ascorbic acid (compounds 1-4) on the phosphorylation-dependent response of stimulated monocytes and neutrophils was investigated. The pyranosyl derivative 3 increased the production of tumor necrosis factor-alpha in human monocytes stimulated with lipopolysaccharide (LPS). Compound 3 also enhanced the release of tumor necrosis factor-alpha from nonstimulated monocytes. Michael adducts 1-4 inhibited the formation of reactive oxygen species in fMLP-stimulated human neutrophils as measured by luminol chemiluminescence. Treatment with 6-O-palmitoyl-L-ascorbic acid (compound 5) also led to a decreased luminescence response of neutrophils. Results are discussed with respect to the inhibitory activity of Michael adducts of ascorbic acids towards protein phosphatases PP1/PP2A.     

Identifying the cause of unilateral hypoperfusion in patients suspected to have chronic pulmonary thromboembolism: diagnostic accuracy of helical CT and conventional angiography

PURPOSE: To determine the prevalence of unilateral hypoperfusion in patients suspected to have chronic thromboembolism (CTE), to identify the most common cause of hypoperfusion, and to compare the accuracy of helical computed tomographic (CT) angiography with that of conventional angiography in helping to determine the cause. MATERIALS AND METHODS: Radionuclide lung scan reports showed asymmetric hypoperfusion in 47 of 410 consecutive patients referred because of suspected CTE. Twenty-seven patients had unilateral or predominantly unilateral perfusion abnormalities. Each pulmonary angiogram and CT angiogram in these patients was interpreted independently by two readers blinded to clinical information and surgical outcome. Surgical confirmation of the diagnosis was available in 39 of the 47 patients with asymmetric hypoperfusion. RESULTS: Unilateral (n = 11) or predominantly unilateral hypoperfusion (n = 16) was found in 6.6% (27 of 410 patients) of patients referred, and CTE was the most common cause. The accuracies of CT angiogram readers (reader 1, 83%; reader 2, 89%) were greater than those of conventional angiogram readers (reader 1, 73%; reader 2, 65%) for distinguishing CTE from other causes. CONCLUSION: Unilateral hypoperfusion occurred in 6.6% of our study population, most frequently because of CTE. CT angiography is an excellent diagnostic alternative to conventional angiography for distinguishing patients with CTE from those with other causes.