Vaccine testing of a recombinant activation-associated secreted protein (ASP1) from Ostertagia ostertagi

Previous vaccination trials against the economically important cattle parasite Ostertagia ostertagi have indicated the protective capacity of activation-associated secreted proteins (ASPs). The further development of these antigens into a commercial vaccine will require their recombinant expression. The aim of the current study was to clone and express Oo-asp1 in a baculovirus expression system and to evaluate the protective capacity of the recombinant protein against an O. ostertagi challenge infection in cattle. The full coding sequence of Oo-asp1 was cloned in a baculovirus expression vector in frame with a carboxy-terminal Histidine tag and recombinant virus was used to infect an insect cell culture. Western blot analysis with anti-His and anti-Oo-ASP1 antibodies showed the production of recombinant Oo-ASP1. The cell pellet containing the recombinant was subsequently used to immunize seven calves three times intramuscularly with QuilA as adjuvant. Control animals were solely injected with the QuilA adjuvant. The challenge infection with O. ostertagi consisted of 30 000 L3 larvae per animal given over 30 days (1000 larvae/day, 5 days/week) and started the same day as the final immunization. Immunization with the recombinant Oo-ASP1 did not result in any level of protection against the challenge infection. There was no reduction in faecal egg output or in worm burdens. Moreover, Western blot analyses and ELISA indicated that, although the animals raised an antibody response against the recombinant Oo-ASP1, there was hardly a response against the native Oo-ASP1, suggesting that the baculovirus expressed recombinant was wrongly folded or lacked essential secondary modifications. Further analysis of the structure of the native ASPs and their glycosylations is being done

Pro-inflammatory properties of stromal cell-derived factor-1 (CXCL12) in collagen-induced arthritis

CXCL12 (stromal cell-derived factor 1) is a unique biological ligand for the chemokine receptor CXCR4. We previously reported that treatment with a specific CXCR4 antagonist, AMD3100, exerts a beneficial effect on the development of collagen-induced arthritis (CIA) in the highly susceptible IFN-γ receptor-deficient (IFN-γR KO) mouse. We concluded that CXCL12 plays a central role in the pathogenesis of CIA in IFN-γR KO mice by promoting delayed type hypersensitivity against the auto-antigen and by interfering with chemotaxis of CXCR4(+ )cells to the inflamed joints. Here, we investigated whether AMD3100 can likewise inhibit CIA in wild-type mice and analysed the underlying mechanism. Parenteral treatment with the drug at the time of onset of arthritis reduced disease incidence and modestly inhibited severity in affected mice. This beneficial effect was associated with reduced serum concentrations of IL-6. AMD3100 did not affect anti-collagen type II antibodies and, in contrast with its action in IFN-γR KO mice, did not inhibit the delayed type hypersensitivity response against collagen type II, suggesting that the beneficial effect cannot be explained by inhibition of humoral or cellular autoimmune responses. AMD3100 inhibited the in vitro chemotactic effect of CXCL12 on splenocytes, as well as in vivo leukocyte infiltration in CXCL12-containing subcutaneous air pouches. We also demonstrate that, in addition to its effect on cell infiltration, CXCL12 potentiates receptor activator of NF-κB ligand-induced osteoclast differentiation from splenocytes and increases the calcium phosphate-resorbing capacity of these osteoclasts, both processes being potently counteracted by AMD3100. Our observations indicate that CXCL12 acts as a pro-inflammatory factor in the pathogenesis of autoimmune arthritis by attracting inflammatory cells to joints and by stimulating the differentiation and activation of osteoclasts

Spring Water Geochemistry: A Geothermal Exploration Tool in the Rhenohercynian Fold-and-Thrust Belt in Belgium

peer reviewedSpring water geochemistry is applied here to evaluate the geothermal potential in Rhenohercynian fold and thrust belt around the deepest borehole in Belgium (Havelange borehole: 5648 m MD). Fifty springs and (few) wells around Havelange borehole were chosen according to a multicriteria approach including the hydrothermal source of “Chaudfontaine” (T ≈ 36 ◦C) taken as a reference for the area. The waters sampled, except Chaudfontaine present an in-situ T range of 3.66–14.04 ◦C (mean 9.83 ◦C) and a TDS (dry residue) salinity range of 46–498 mg/L. The processing methods applied to the results are: hierarchical clustering, Piper and Stiff diagrams, TIS, heat map, boxplots, and geothermometry. Seven clusters are found and allow us to define three main water types. The first type, locally called “pouhon”, is rich in Fe and Mn. The second type contains an interesting concentration of the geothermal indicators: Li, Sr, Rb. Chaudfontaine and Moressée (≈5 km East from the borehole) belong to this group. This last locality is identified as a geothermal target for further investigations. The third group represents superficial waters with frequently high NO3 concentration. The application of conventional geothermometers in this context indicates very different reservoir temperatures. The field of applications of these geothermometers need to be review in these geological conditions.MEE

The transcriptional co-activator LEDGF/p75 displays a dynamic scan-and-lock mechanism for chromatin tethering

Nearly all cellular and disease related functions of the transcriptional co-activator lens epithelium-derived growth factor (LEDGF/p75) involve tethering of interaction partners to chromatin via its conserved integrase binding domain (IBD), but little is known about the mechanism of in vivo chromatin binding and tethering. In this work we studied LEDGF/p75 in real-time in living HeLa cells combining different quantitative fluorescence techniques: spot fluorescence recovery after photobleaching (sFRAP) and half-nucleus fluorescence recovery after photobleaching (hnFRAP), continuous photobleaching, fluorescence correlation spectroscopy (FCS) and an improved FCS method to study diffusion dependence of chromatin binding, tunable focus FCS. LEDGF/p75 moves about in nuclei of living cells in a chromatin hopping/scanning mode typical for transcription factors. The PWWP domain of LEDGF/p75 is necessary, but not sufficient for in vivo chromatin binding. After interaction with HIV-1 integrase via its IBD, a general protein–protein interaction motif, kinetics of LEDGF/p75 shift to 75-fold larger affinity for chromatin. The PWWP is crucial for locking the complex on chromatin. We propose a scan-and-lock model for LEDGF/p75, unifying paradoxical notions of transcriptional co-activation and lentiviral integration targeting