Characterization of Schistosome Tegumental Alkaline Phosphatase (SmAP)

Schistosomes are parasitic platyhelminths that currently infect over 200 million people globally. The parasites can live for years in a putatively hostile environment - the blood of vertebrates. We have hypothesized that the unusual schistosome tegument (outer-covering) plays a role in protecting parasites in the blood; by impeding host immunological signaling pathways we suggest that tegumental molecules help create an immunologically privileged environment for schistosomes. In this work, we clone and characterize a schistosome alkaline phosphatase (SmAP), a predicted ∼60 kDa glycoprotein that has high sequence conservation with members of the alkaline phosphatase protein family. The SmAP gene is most highly expressed in intravascular parasite life stages. Using immunofluorescence and immuno-electron microscopy, we confirm that SmAP is expressed at the host/parasite interface and in internal tissues. The ability of living parasites to cleave exogenous adenosine monophosphate (AMP) and generate adenosine is very largely abolished when SmAP gene expression is suppressed following RNAi treatment targeting the gene. These results lend support to the hypothesis that schistosome surface enzymes such as SmAP could dampen host immune responses against the parasites by generating immunosuppressants such as adenosine to promote their survival. This notion does not rule out other potential functions for the adenosine generated e.g. in parasite nutrition

Transcriptional Changes in Schistosoma mansoni during Early Schistosomula Development and in the Presence of Erythrocytes

Schistosome blood flukes cause more mortality and morbidity than any other human worm infection, but current control methods primarily rely on a single drug. There is a desperate need for new approaches to control this parasite, including vaccines. People become infected when the free-swimming larva, the cercaria, enters through the skin and becomes the schistosomulum. Schistosomula are susceptible to immune responses during their first few days in the host before they become adult parasites. We characterised the genes that these newly transformed parasites switch on when they enter the host to identify molecules that are critical for survival in the human host. Some of these highly up-regulated genes can be targeted for future development of new vaccines and drugs

Stainless steel weld metal designed to mitigate residual stresses

There have been considerable efforts to create welding consumables which on solid state phase transformation partly compensate for the stresses which develop when a constrained weld cools to ambient temperatures. All of these efforts have focused on structural steels which are ferritic. In the present work, alloy design methods have been used to create a stainless steel welding consumable which solidifies as δ ferrite, transforms almost entirely into austenite which then undergoes martensitic transformation at a low temperature of about 220◦C. At the same time, the carbon concentration has been kept to a minimum to avoid phenomena such as sensitisation. The measured mechanical properties, especially toughness, seem to be significantly better than commercially available martensitic stainless steel welding consumables, and it has been demonstrated that the use of the new alloy reduces distortion in the final joint

A Multicentric, Open-Label, Randomized, Comparative Clinical Trial of Two Different Doses of Expanded hBM-MSCs Plus Biomaterial versus Iliac Crest Autograft, for Bone Healing in Nonunions after Long Bone Fractures: Study Protocol

ORTHOUNION is a multicentre, open, comparative, three-arm, randomized clinical trial (EudraCT number 2015-000431-32) to compare the efficacy, at one and two years, of autologous human bone marrow-derived expanded mesenchymal stromal cell (hBM-MSC) treatments versus iliac crest autograft (ICA) to enhance bone healing in patients with diaphyseal and/or metaphysodiaphyseal fracture (femur, tibia, and humerus) status of atrophic or oligotrophic nonunion (more than 9 months after the acute fracture, including recalcitrant cases after failed treatments). The primary objective is to determine if the treatment with hBM-MSCs combined with biomaterial is superior to ICA in obtaining bone healing. If confirmed, a secondary objective is set to determine if the dose of 100 × 106 hBM-MSCs is noninferior to that of 200 × 106 hBM-MSCs. The participants (n = 108) will be randomly assigned to either the experimental low dose (n = 36), the experimental high dose (n = 36), or the comparator arm (n = 36) using a central randomization service. The trial will be conducted in 20 clinical centres in Spain, France, Germany, and Italy under the same clinical protocol. The confirmation of superiority for the proposed ATMP in nonunions may foster the future of bone regenerative medicine in this indication. On the contrary, absence of superiority may underline its limitations in clinical use

Surface proteins and antigens of adult Schistosoma mansoni tegumental membranes detached onto poly-lysine coated beads

Poly-lysine coated beads attached readily onto Schistosoma mansoni. On detachment, the beads removed membranes from the surface of the tegument. Analysis of the proteins of the detached membranes showed that three major proteins of 94, 73 and 62 kDa were present in contrast to a more complex range of proteins present in the phosphate-buffered saline released membranes. The membranes attached to beads were radio-iodinated and the antigens examined in immunoprecipitates by sodium dodecyl sulphate-polyacrylamide gel electrophoresis using various antisera. In addition to the well-established 32 and 20 kDa antigens of the tegument, other major antigens of 200, 25 and 11-12 kDa were iodinated in the membranes attached to the beads. The results suggest that the major antigens studied in the tegument may not correspond to the major proteins identified. The present approach shows promise for deducing the topography of the surface antigens and proteins of schistosomes

The phospholipid and fatty acid composition of Schistosoma mansoni and of its purified tegumental membranes

The lipid compositions of mature male and female Schistosoma mansoni and cercariae were compared to that of the hepato-pancreas of unparasitised Biomphalaria globrata (the intermediate snail host), and of red blood cells and sera of hamsters (the mammalian host). Membranes were isolated from the tegument of mature schistosomes by spontaneous release into phosphate-buffered saline, with or without vortexing, and by removal from the parasite's surface using poly-lysine beads. The phospholipid composition of the membranes prepared by the three methods showed a typical plasma membrane-like profile, with high sphingomyelin content (approximately 20%) and cholesterol to phospholipid molar ratio (0.7-1.1). The fatty acid compositions of the resolved phospholipid classes were analysed. Although the composition was in general unremarkable, a high content of eicosaenoic acid (20:1), rarely found in mammals, was noted in whole schistosomes, cercariae, the hepato-pancreas of unparasitised Biomphalaria, and the isolated tegumental membranes. Eicosaenoic acid was also found in adults of Schistosoma japonicum. Host serum lipids from normal and parasitised hamsters contained extremely low amounts of eicosaenoic acid, indicating that this fatty acid is probably continually synthesised by the parasite, probably from preformed fatty acid precursors provided by the host

Purification and topographical location of tegumental alkaline phosphatase from adult Schistosoma mansoni

Alkaline phosphatase, a marker for tegumental membranes of Schistosoma mansoni, was extracted using Triton X-100 from membranes purified by sucrose density gradient centrifugation. The enzyme activity was purified 6 800-fold over parasite homogenates and 118-fold over the tegumental membranes released when parasites were incubated in phosphate-buffered saline. Purification of the solubilised enzyme was achieved by binding to a Con A agarose affinity column, gel filtration of the eluted glycoproteins, and Blue affigel chromatography. The purified enzyme was shown to consist of a single glycosylated polypeptide Mr 65 000 on reduced sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Enzyme activity was associated with a possible tetramer, Mr 260 000 on gel filtration. Activity was associated with a band Mr 130 000 in sodium dodecyl sulphate-polyacrylamide gel electrophoresis run in the absence of reducing agents. Using sodium dodecyl sulphate-polyacrylamide gel electrophoresis in the absence of reducing agents, the size of the enzyme was shown to be similar in cercariae, schistosomula, adult schistosomes and their eggs, but it was smaller than the activity (Mr 145 000) extracted from host liver and intestinal microsomal membranes. The topography of the enzyme in the schistosome tegument was investigated by using surface radio-labelling reagents followed by its purification. The enzyme could be radio-iodinated only with difficulty in adult worms. Bolton and Hunter reagent, used at high concentration and for prolonged time periods, resulted in labelling of enzyme activity and the Mr 65 000 polypeptide subunit was also iodinated under these extreme conditions. It was concluded that the enzyme is not exposed at the schistome's surface, and is probably buried in the tegumental membrane network

Glycosyl transferase activities are associated with the surface membrane in adult Schistosoma mansoni

Incubation of live adult Schistosoma mansoni in a variety of media released tegumental material containing membrane bound alkaline phosphatase, mannosyl transferase and galactosyl transferase activities. Centrifugation of the tegumental material released by incubation of worms in phosphate-buffered saline in sucrose density gradients yielded a pellet and four fractions, two of which consisted mainly of surface membranes. The distribution of the enzymes in the gradient, espeically in the two surface membrane-containing subfractions was similar. Application of the "digitonin shift" technique showed that the membranes containing the enzyme reactivities were moved to an equal extent into a denser part of the sucrose gradient. Thus the enzymes are located on the same or similar cholesterol-containing membranes. It is concluded that the transferases, like the alkaline phosphatases, are located in the surface membranes of S. mansoni and the consequences of this location for the host-parasite interaction are discussed