Nigel B. Rendell

Specific C-Terminal Cleavage and Inactivation of Interleukin-8 by Invasive Disease Isolates of Streptococcus pyogenes

Lethal necrotizing fasciitis caused by Streptococcus pyogenes is characterized by a paucity of neutrophils at the site of infection. Interleukin (IL)-8, which is important for neutrophil transmigration and activation, can be degraded by S. pyogenes. Blood isolates of S. pyogenes were better able to degrade human IL-8 than throat isolates. Degradation of IL-8 was the result of a single specific cleavage between 59glutamine and 60arginine within the IL-8 C-terminal alpha helix. Cleaved IL-8 reduced neutrophil activation and migration. IL-8-cleaving activity was found in partially purified supernatant of a necrotizing fasciitis isolate, and this activity was associated with an approximately 150-kDa fraction containing S. pyogenes cell envelope proteinase (SpyCEP). IL-8-cleaving activity corresponded with the presence of SpyCEP in the supernatant. Cleavage of IL-8 by S. pyogenes represents an unprecedented mechanism of immune evasion, effectively preventing IL-8 C-terminus-mediated endothelial translocation and subsequent recruitment of neutrophils.

Plasma mevalonic acid, an index of cholesterol synthesis in vivo, and responsiveness to HMG-CoA reductase inhibitors in familial hypercholesterolaemia

Fasting plasma mevalonic acid (MVA), an indicator of in vivo cholesterol synthesis, was measured in 35 patients with familial hypercholesterolaemia (FH) of whom 7 were treated with pravastatin 10-40 mg/day, 7 with simvastatin 10-40 mg/day and 21 with atorvastatin 80 mg/day. Reductions in low density lipoprotein (LDL) cholesterol and MVA on maximal dose therapy differed significantly between the three drugs: 34.7%, 42.9% and 54.0% (P = 0.0001), and 31.6%, 48.9% and 58.8% (P = 0.004), respectively. Patients on atorvastatin were subdivided according to whether their reduction in LDL cholesterol on treatment was above or below the mean percentage change for the whole group. Basal values of LDL cholesterol did not differ significantly, but above average responders had a significantly higher mean pre-treatment level of MVA (6.2 +/- 0.60 vs. 4.3 +/- 0.61 ng/ml, P < 0.05) than below average responders. When all three drug groups were pooled above average responders showed a significantly greater absolute decrease in MVA on treatment than below average responders (3.85 +/- 0.48 vs. 2.33 +/- 0.40 ng/ml, P < 0.05). However, there was no significant correlation between the magnitude of the decreases in LDL cholesterol and MVA. These findings suggest that FH patients who responded well to statins had a higher basal level of plasma MVA, i.e. a higher rate of cholesterol synthesis, which was more susceptible to pharmacological inhibition. The more marked cholesterol lowering effect of atorvastatin 80 mg/day presumably reflects, at least in part, its ability to inhibit HMG-CoA reductase to a greater extent than maximal recommended doses of pravastatin and simvastatin of 40 mg/day.

Characterisation of Pseudomonas rhamnolipids

The Gram negative organism, Pseudomonas aeruginosa, is often found in the lungs of patients with cystic fibrosis and other forms of severe bronchiectasis, where it secretes a number of extracellular toxins including the mono- and dirhamnolipids. The principal monorhamnolipid from P. aeruginosa has previously been identified as rhamnosyl-3-hydroxydecanoyl-3-hydroxydecanoate (Rh-C10.C10). A number of related mono- and dirhamnolipids have been purified from cultures of a clinical isolate of P. aeruginosa and identified by fast atom bombardment and electron impact mass spectrometry: these contain the 3-hydroxyoctanoyl-3-hydroxydecanoate (C8.C10) and 3-hydroxydecanoyl-3-hydroxydodecanoate (C10.C12) homologues. Structural isomers were also present where the order of the lipid linkage was transposed (Rh-C10.C8 and Rh-C12.C10). Unsaturated mono- and dirhamnolipids containing the 3-hydroxydecanoyl-3-hydroxydodec-5-enoate (C10.C12:1) lipid were also present.

Comparison of the effects of dietary plant sterol and stanol esters on lipid metabolism

BACKGROUND AND AIM To compare the cholesterol-lowering efficacy and other metabolic effects of plant sterol and stanol esters, both of which are commonly used in the dietary management of hypercholesterolaemia. METHODS AND RESULTS The cholesterol-lowering efficacy of equivalent intakes of sterol and stanol esters and of different intakes of stanol esters were compared at 1 and 2 months, both in normal subjects and treated patients with familial hypercholesterolaemia. Systemic effects were assessed by measuring serum levels of plant sterols and of lathosterol and 7alpha-hydroxy-cholestenone, indices of sterol absorption and of cholesterol and bile acid synthesis respectively. There were no significant differences during the study between 1.6g daily of sterol and stanol esters in reducing total cholesterol (by 3-7%) or low density lipoprotein cholesterol (by 4-8%), nor between 1.6 and 2.6 g daily of stanol. However, the cholesterol-lowering effect of plant sterol esters was attenuated between 1 and 2 months. This was accompanied by increased serum plant sterols and decreased levels of 7alpha-hydroxy-cholestenone, especially in statin-treated hypercholesterolaemic patients not taking bile acid sequestrants. CONCLUSIONS These findings suggest that absorption of dietary plant sterols suppressed bile acid synthesis, thereby diminishing their cholesterol-lowering efficacy. In contrast, plant stanols reduced plant sterol absorption and maintained their cholesterol-lowering efficacy.

Reduction by inhibitors of mono(ADP-ribosyl)transferase of chemotaxis in human neutrophil leucocytes by inhibition of the assembly of filamentous actin.

1 Chemotaxis of human neutrophils is mediated by numerous agents [e.g. N‐formyl‐methionyl‐leucyl‐phenylalanine (FMLP) and platelet activating factor (PAF)] whose receptors are coupled to phospholipase C. However, the subsequent transduction pathway mediating cell movement remains obscure. We now propose involvement of mono(ADP‐ribosyl)transferase activity in receptor‐dependent chemotaxis. 2 Human neutrophils were isolated from whole blood and measurements were made of FMLP or PAF‐dependent actin polymerization and chemotaxis. The activity of cell surface Arg‐specific mono(ADP‐ribosyl)transferase was also measured. Each of these activities was inhibited by vitamin K3 and similar IC50 values obtained (4.67 ± 1.46 μm, 2.0 ± 0.1 μm and 4.7 ± 0.1 μm respectively). 3 There were similar close correlations between inhibition of (a) enzyme activity and (b) actin polymerization or chemotaxis by other known inhibitors of mono(ADP‐ribosyl)transferase, namely vitamin K1 novobiocin, nicotinamide and the efficient pseudosubstrate, diethylamino(benzylidineamino)guanidine (DEA‐BAG). 4 Intracellular Ca2+ was measured by laser scanning confocal microscopy with two fluorescent dyes (Fluo‐3 and Fura‐Red). Exposure of human neutrophils to FMLP or PAF was followed by transient increases in intracellular Ca2+ concentration, but the inhibitors of mono(ADP‐ribosyl)transferase listed above had no effect on the magnitude of the response. 5 A panel of selective inhibitors of protein kinase C, tyrosine kinase, protein kinases A and G or phosphatases 1 and 2A showed no consistent inhibition of FMLP‐dependent polymerization of actin. 6 We conclude that eukaryotic Arg‐specific mono(ADP‐ribosyl)transferase activity may be implicated in the transduction pathway mediating chemotaxis of human neutrophils, with involvement in the assembly of actin‐containing cytoskeletal microfilaments.

Acute hyperinsulinaemia decreases cholesterol synthesis less in subjects with non‐insulin‐dependent diabetes mellitus than in non‐diabetic subjects

To investigate the effect of insulin on cholesterol synthesis in vivo we measured plasma mevalonic acid (MVA) concentrations using gas chromatography–mass spectrometry in six non‐obese patients with non‐insulin‐dependent diabetes mellitus (NIDDM) [four men, two women; age 57.5±2.2 years (mean±SEM); glycated haemoglobin (HbA1) 8.5±0.5%; total cholesterol (TC) 5.7±0.5 mmol L−1, triglyceride (TG) 3.8±0.9 mmol L−1] and six non‐diabetic, sex‐ and age‐matched control subjects (age 55.7±2.8 years; HbA1 6.5±0.1%; TC 5.4±0.3 mmol L−1, TG 1.2±0.1 mmol L−1). Subjects were studied twice: during 13‐h hyperinsulinaemic (1 mu kg−1 min−1), euglycaemic (5 mmol L−1) clamp and during a saline infusion. Baseline MVA concentration was significantly higher in diabetic patients than in control subjects (9.8±0.7 ng mL−1 vs. 5.6±0.9 ng mL−1P=0.004). At the end of each study, MVA concentration, expressed as a percentage of baseline, was significantly lower during the hyperinsulinaemic, euglycaemic clamp than during the saline study in both the diabetic (54.4±5.3% vs. 69.6±6.3%, P=0.036) and control subjects (30.5±3.4% vs. 61.7±6.0%, P=0.01). However, the decrease in MVA during the hyperinsulinaemic clamp study was more marked in the control subjects than in the diabetic subjects (P=0.03). A significant positive correlation was found between percentage decrease of MVA and non‐esterified fatty acids following the insulin clamp in NIDDM (r=0.83, P=0.04). We conclude that acute hyperinsulinaemia decreases cholesterol synthesis less in subjects with NIDDM than in non‐diabetic subjects and that this phenomenon, together with increased basal cholesterol synthesis in NIDDM, may in part be due to insulin resistance.

Upregulation of cholesterol synthesis after acute reduction of low density lipoprotein by apheresis in normocholesterolaemic subjects : Evidence for a threshold effect

The influence of low density lipoproteins (LDL) in the plasma on the regulation of cholesterol biosynthesis is not clear. We studied the changes in plasma mevalonic acid (MVA) concentration and the lathosterol/cholesterol (L/C) ratio, which are well established indices of whole body cholesterol synthesis, in four normocholesterolaemic subjects after each had undergone LDL apheresis on two occasions. LDL apheresis of 75% of the calculated plasma volume reduced LDL-cholesterol by 44% to 1.5 +/- 0.2 mmol/l without changing plasma MVA levels or L/C ratios. Apheresis of 125% of the calculated plasma volume decreased plasma LDL-cholesterol by 69% to 0.9 +/- 0.2 mmol/l, with significant increases in plasma MVA and L/C ratio on the day after the procedure. These results imply that LDL-cholesterol is an integral part of the sterol regulatory pool and suggest that plasma levels cannot be lowered below 1-1.4 mmol/l in normal subjects without upregulating cholesterol biosynthesis.

Formation and metabolism of 14,15-epoxyeicosatrienoic acid by human reproductive tissues

Human granulosa-luteal cells cultured in the presence of arachidonic acid produced low levels of the epoxygenase metabolite 14,15-epoxy-5,8,11-(Z,Z,Z)-eicosatrienoic acid (14,15-EpETrE) as determined by HPLC analysis and gas chromatography mass spectrometry. When authentic 14,15-[3H]EpETrE was incubated with these cells in the absence of serum it was metabolised initially to the dihydroxy derivative (14,15-dihydroxy-5,8,11-eicosatrienoic acid, 14,15-DiHETrE) and subsequently to a number of more polar metabolites as determined by HPLC. Fetal calf serum protected 14,15-EpETrE from metabolism for at least 2 h. A similar pattern of metabolism was obtained when 14,15-[3H]EpETrE was incubated with a human choriocarcinoma cell line (BeWo). Microsomes from this cell line converted arachidonic acid to a large number of radioactive metabolites including 14,15-DiHETrE and 11,12-DiHETrE although there was no evidence for the parent epoxides. These results extend earlier findings that human reproductive tissues produce epoxygenase metabolites, and demonstrate the rapid metabolism of these compounds by intact cells in the absence of serum.

Metabolism of prostaglandins E2 and F2α by human fetal membranes

Prostaglandin E2 (PGE2) is important in the early stages of human labour, leading particularly to cervical ripening and dilatation. The source of PGE2 is thought to be either the amnion or the decidua, but the chorion interposes between the amnion and the target tissues, namely the myometrium and cervix. In order to investigate the role of the chorion in modulating prostanoid production, [3H]PGE2 was added to the amnion side of fetal membranes, and the production of metabolites on both sides of the fetal membrane followed by HPLC. The major metabolite was 13,14-dihydro-15-oxo-PGE2 with smaller amounts of 13,14-dihydro-15-oxo-PGA2 and PGB2. The production of all metabolites of PGE2 was time dependent. [3H]PGF2 alpha, which is normally produced by the decidua, was also added to fetal membranes and found to be metabolised to 13,14-dihydro-15-oxo-PGF2 alpha and PGE2. These results suggest that the metabolic enzymes in the chorion may determine intra-uterine levels of prostaglandins, and may also determine the identity of the eicosanoids released by intact fetal membranes.

Two types of amyloid in a single heart.

To the editor: Amyloidosis is a heterogeneous group of diseases, in which amyloidogenic precursor proteins misfold and adopt a β-pleated sheet conformation.[1][1] Several proteins can form amyloid fibrils in vivo including transthyretin,[2][2] apolipoprotein A-I and A-II, lysozyme, fibrinogen,