The obligate intracellular pathogen Leishmania major survives and multiplies in professional phagocytes. The evasion strategy to circumvent killing by host phagocytes and establish a productive infection is poorly understood. Here we report that the virulent inoculum of Leishmania promastigotes contains a high ratio of annexin A5-binding apoptotic parasites. This subpopulation of parasites is characterized by a round body shape, a swollen kinetoplast, nuclear condensation, and a lack of multiplication and represents dying or already dead parasites. After depleting the apoptotic parasites from a virulent population, Leishmania do not survive in phagocytes in vitro and lose their disease-inducing ability in vivo . TGF-β induced by apoptotic parasites is likely to mediate the silencing of phagocytes and lead to survival of infectious Leishmania populations. The data demonstrate that apoptotic promastigotes, in an altruistic way, enable the intracellular survival of the viable parasites.

Considering the high incidence of galactofuranose (Gal f ) in pathogens and its absence from higher eukaryotes, the enzymes involved in the biosynthesis of this unusual monosaccharide appear as attractive drug targets. However, although the importance of Gal f in bacterial survival or pathogenesis is established, its role in eukaryotic pathogens is still undefined. Recently, we reported the identification and characterization of the first eukaryotic UDP-galactopyranose mutases. This enzyme holds a central role in Gal f metabolism by providing UDP-Gal f to all galactofuranosyltransferases. In this work, the therapeutical potential of Gal f metabolism in Leishmania major was hence evaluated by targeted replacement of the GLF gene encoding UDP-galactopyranose mutase. In L. major, Gal f is present in the membrane anchor of the lipophosphoglycan (LPG) and in glycoinositolphospholipids. Accordingly, the generated glf ؊ mutant is deficient in LPG backbone and expresses truncated glycoinositolphospholipids. These structural changes do not influence the in vitro growth of the parasite but lead to an attenuation of virulence comparable with that observed with a mutant exclusively deficient in LPG.

The nucleotide sugar UDP-galactose (UDP-Gal) is essential for the biosynthesis of several abundant glycoconjugates forming the surface glycocalyx of the protozoan parasite Leishmania major. Current data suggest that UDP-Gal could arise de novo by epimerization of UDP-glucose (UDP-Glc) or by a salvage pathway involving phosphorylation of Gal and the action of UDP-glucose:alpha-D-galactose-1-phosphate uridylyltransferase as described by Leloir. Since both pathways require UDP-Glc, inactivation of the UDP-glucose pyrophosphorylase (UGP) catalyzing activation of glucose-1 phosphate to UDP-Glc was expected to deprive parasites of UDP-Gal required for Leishmania glycocalyx formation. Targeted deletion of the gene encoding UGP, however, only partially affected the synthesis of the Gal-rich phosphoglycans. Moreover, no alteration in the abundant Gal-containing glycoinositolphospholipids was found in the deletion mutant. Consistent with these findings, the virulence of the UGP-deficient mutant was only modestly affected. These data suggest that Leishmania elaborates a UDP-Glc independent salvage pathway for UDP-Gal biosynthesis.

The lipoxin A4 receptor (ALX) is an important target of LxA4 in synovial tissues of patients with inflammatory arthritis. Previously this receptor was known as the FPRL-1 on PMN and shown to interact with acute phase proteins and a variety of peptides. ALX signalling can either activate or deactivate PMN functions. In this study, we found that both LxA4 and a chemotactic lipid leishmania chemotactic factor released by the parasite leishmania increased infectivity of this pathogen in an ALX dependent fashion. This functional characterization of ALX could lead to development of novel, therapeutic targets for treatment inflammatory diseases.

The influence of the xanthine derivative pentoxifylline ('Trental' or BL191; Hoechst-Roussel) on exercise tolerance was measured in 38 subjects with stable, severe to moderately severe, intermittent claudication who completed a randomised, double-blind, placebo controlled, cross-over clinical trial. Patients received placebo tablets or 400 mg slow-release pentoxifylline tablets ('Trental 400') twice a day for one week, followed by three times daily for seven weeks, and then crossed over to receive the alternate preparation for another eight weeks. Claudication distance and walking distance were measured on a treadmill before starting treatment and again at four-week intervals during the trial. At the same times, red blood cell filterability, plasma fibrinogen concentration and blood viscosity, resting and post-ischemic calf muscle blood flow, and the resting and post-exercise ankle/brachial systolic pressure ratio were also measured. In this study, the observed effects of pentoxifylline treatment were no greater than those of placebo, even though serum levels of pentoxifylline and its hydroxy-metabolite were within the anticipated range. This was shown by a 'therapeutic effect ratio' of 0.98 for treadmill claudication distance and 0.96 for treadmill walking distance after within-patient analysis at the end of the cross-over (where a ratio of 1.0 means the test drug and placebo effects are identical). These ratios have 95% confidence limits of 0.72-1.34 and 0.74-1.25, respectively.

We have used the human ECV 304 cell line to study the origin and fate of extracellular RNA (exRNA) in cell culture. Quantification of different extracellular RNA species using reverse transcription followed by quantitative PCR revealed a prevalent fraction of ribosomal RNAs. Comparison of intracellular and extracellular ribosomal RNA copy numbers allowed the calculation of the number of destroyed cells that would result in the corresponding number of extracellular rRNAs. Interestingly, this number was comparable to the amount of destroyed cells as determined by the measurement of extracellular lactate dehydrogenase activity.

SUMMARY.Erythrocytes maintained in vitro under controlled conditions at pH 7·40 were unable to respond to changes in extracellular phosphate concentrations with changes in intracellular 2,3-bisphosphoglycerate content over an 8 h incubation period. Increased extracellular phosphate concentrations did not retard the rate at which 2,3-bisphosphoglycerate concentrations fell when erythrocytes were incubated at pH 7·10. Elevated extracellular phosphate concentrations did not increase the rate of recovery of intracellular 2,3-bisphosphoglycerate concentrations when erythrocytes were returned to pH 7·40 having been incubated at pH 7·10. The rate at which 2,3-bisphosphoglycerate concentrations increased under these latter conditions was too slow to counterbalance any effect that adjustment of blood pH may have on the position of the haemoglobin-oxygen dissociation curve during treatment of diabetic ketoacidosis.