Chemokine secretion of rheumatoid arthritis synovial fibroblasts stimulated by Toll-like receptor 2 ligands

To analyze the role of Toll-like receptors (TLR) in the pathogenesis of rheumatoid arthritis, we have assessed the effects of stimulation of cultured synovial fibroblasts by the TLR-2 ligand bacterial peptidoglycan. By using high density oligonucleotide microarray analysis we identified 74 genes that were up-regulated >2.5-fold. Fourteen CC and CXC chemokine genes were among the genes with the highest up-regulation. Quantitative real-time PCR analysis confirmed up-regulation of granulocyte chemotactic protein (GCP)-2, RANTES, monocyte chemoattractant protein (MCP)-2, IL-8, growth-related oncogene-2, and to a lesser extent, macrophage-inflammatory protein 1alpha, MCP-1, EXODUS, and CXCL-16. GCP-2, RANTES, and MCP-2 were detected in culture supernatants of synovial fibroblasts stimulated with peptidoglycan. Chemokine secretion induced by stimulation of rheumatoid arthritis synovial fibroblasts via TLR-2 was functionally relevant as demonstrated by chemotaxis assays. GCP-2 and MCP-2 expression, which have not been reported previously in rheumatoid arthritis, was demonstrated in synovial tissue sections of patients diagnosed with rheumatoid arthritis but not in those with osteoarthritis. Correspondingly, synovial fluid levels were significantly higher in patients diagnosed with rheumatoid arthritis as compared with osteoarthritis. Thus, we present evidence for an induction of chemokine secretion by activation of synovial fibroblasts via TLR-2, possibly contributing to the formation of inflammatory infiltrates characteristically found in rheumatoid arthritis joints.     

An Na+-pumping V1V0-ATPase complex in the thermophilic bacterium Clostridium fervidus.

Energy transduction in the anaerobic, thermophilic bacterium Clostridium fervidus relies exclusively on Na+ as the coupling ion. The Na+ ion gradient across the membrane is generated by a membrane-bound ATPase (G. Speelmans, B. Poolman, T. Abee, and W. N. Konings, J. Bacteriol. 176:5160-5162, 1994). The Na+-ATPase complex was purified to homogeneity. It migrates as a single band in native polyacrylamide gel electrophoresis and catalyzes Na+-stimulated ATPase activity. Denaturing gel electrophoresis showed that the complex consists of at least six different polypeptides with apparent molecular sizes of 66, 61, 51, 37, 26, and 17 kDa. The N-terminal sequences of the 66- and 51-kDa subunits were found to be significantly homologous to subunits A and B, respectively, of the Na+-translocating V-type ATPase of Enterococcus hirae. The purified V1V0 protein complex was reconstituted in a mixture of Escherichia coli phosphatidylethanolamine and egg yolk phosphatidylcholine and shown to catalyze the uptake of Na+ ions upon hydrolysis of ATP. Na+ transport was completely abolished by monensin, whereas valinomycin stimulated the uptake rate. This is indicative of electrogenic sodium transport. The presence of the protonophore SF6847 had no significant effect on the uptake, indicating that Na+ translocation is a primary event and in the cell is not accomplished by an H+-translocating pump in combination with an Na+-H+ antiporter.     

De <Emphasis Type="Italic">BPSD-DS</Emphasis> evaluatieschaal voor dementiegerelateerde gedragsveranderingen bij mensen met downsyndroom

Behavioral and psychological symptoms of dementia (BPSD) have not been comprehensively studied in people with Down syndrome, despite their high risk on dementia. A novel evaluation scale was developed to identify the nature, frequency and severity of behavioral changes (83 behavioral items in 12 clinically defined sections). Central aim was to identify items that change in relation to the dementia status. Structured interviews were conducted with informants of people with Down syndrome without dementia (DS, N?=?149), with questionable dementia (DS?+?TD, N?=?65) and with diagnosed dementia (DS?+?AD, N?=?67). Group comparisons showed a pronounced increase in frequency and severity of items about anxiety, sleep disturbances, agitation & stereotypical behavior, aggression, apathy, depressive symptoms, and, eating/drinking behavior. The proportion of individuals presenting an increase was highest in the DS?+?AD group and lowest in the DS group. Interestingly, among DS?+?TD individuals, a substantial proportion already presented increased anxiety, sleep disturbances, apathy and depressive symptoms, suggesting that these changes may be early alarm signals of dementia. The scale may contribute to a better understanding of the changes, adapting daily care/support, and providing suitable therapies to people with Down syndrome. The scale needs to be optimized based on the results and experiences. The applicability, reliability and validity require further study.     

Identifying Malaria Transmission Foci for Elimination Using Human Mobility Data

Humans move frequently and tend to carry parasites among areas with endemic malaria and into areas where local transmission is unsustainable. Human-mediated parasite mobility can thus sustain parasite populations in areas where they would otherwise be absent. Data describing human mobility and malaria epidemiology can help classify landscapes into parasite demographic sources and sinks, ecological concepts that have parallels in malaria control discussions of transmission foci. By linking transmission to parasite flow, it is possible to stratify landscapes for malaria control and elimination, as sources are disproportionately important to the regional persistence of malaria parasites. Here, we identify putative malaria sources and sinks for pre-elimination Namibia using malaria parasite rate (PR) maps and call data records from mobile phones, using a steady-state analysis of a malaria transmission model to infer where infections most likely occurred. We also examined how the landscape of transmission and burden changed from the pre-elimination setting by comparing the location and extent of predicted pre-elimination transmission foci with modeled incidence for 2009. This comparison suggests that while transmission was spatially focal pre-elimination, the spatial distribution of cases changed as burden declined. The changing spatial distribution of burden could be due to importation, with cases focused around importation hotspots, or due to heterogeneous application of elimination effort. While this framework is an important step towards understanding progressive changes in malaria distribution and the role of subnational transmission dynamics in a policy-relevant way, future work should account for international parasite movement, utilize real time surveillance data, and relax the steady state assumption required by the presented model.     

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines¹ support our understanding of invasion of the uterine wall² and remodeling of uterine spiral arteries^3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts^5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation^7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies^10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu¹³.We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues^7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models^10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.     

Gαq-mediated plasma membrane translocation of sphingosine kinase-1 and cross-activation of S1P receptors

Sphingosine-1-phosphate (S1P), formed by sphingosine kinases (SphKs), regulates cellular proliferation and migration by acting as an agonist at specific receptors or intracellularly. Since S1P's effects are probably dependent on subcellular localization of its formation and degradation, we have studied the influence of G protein-coupled receptors on the localization of SphK1. Activation of G_q-coupled receptors induced a profound, rapid (half-life 3–5 s) and long-lasting (> 2 h) translocation of SphK1 to the plasma membrane. This was mimicked by expression of constitutively active G protein α-subunits specifically of the G_q family. Classical G_q signalling pathways, or phosphorylation at Ser²²⁵, phospholipase D and Ca²⁺/calmodulin were not involved in M₃ receptor-induced SphK1 translocation in HEK-293 cells. Translocation was associated with S1P receptor internalization, which was dependent on catalytic activity of SphK1 and S1P receptor binding and thus resulted from S1P receptor cross-activation. It is concluded that SphK1 is an important effector of G_q-coupled receptors, linking them via cross-activation of S1P receptors to G_i and G_12/13 signalling pathways.     

More reliable estimates of divergence times in Pan using complete mtDNA sequences and accounting for population structure

Here, we report the sequencing and analysis of eight complete mitochondrial genomes of chimpanzees (Pan troglodytes) from each of the three established subspecies (P. t. troglodytes, P. t. schweinfurthii and P. t. verus) and the proposed fourth subspecies (P. t. ellioti). Our population genetic analyses are consistent with neutral patterns of evolution that have been shaped by demography. The high levels of mtDNA diversity in western chimpanzees are unlike those seen at nuclear loci, which may reflect a demographic history of greater female to male effective population sizes possibly owing to the characteristics of the founding population. By using relaxed-clock methods, we have inferred a timetree of chimpanzee species and subspecies. The absolute divergence times vary based on the methods and calibration used, but relative divergence times show extensive uniformity. Overall, mtDNA produces consistently older times than those known from nuclear markers, a discrepancy that is reduced significantly by explicitly accounting for chimpanzee population structures in time estimation. Assuming the human–chimpanzee split to be between 7 and 5 Ma, chimpanzee time estimates are 2.1–1.5, 1.1–0.76 and 0.25–0.18 Ma for the chimpanzee/bonobo, western/(eastern + central) and eastern/central chimpanzee divergences, respectively.     

Quantifying the historic contribution of Olympia oysters to filtration in Pacific Coast (USA) estuaries and the implications for restoration objectives

The Olympia oyster, Ostrea lurida Carpenter, was formerly widespread in many US Pacific coast estuaries. Following dramatic declines in the late 1800s and early 1900s, this species is now the focus of renewed restoration efforts. Restoration is undertaken for brood stock rehabilitation as well as a range of ecosystem services such as filtration; however, these ecosystem services are as yet poorly quantified. We present the first laboratory measurements of filtration rates (FR) for O. lurida, to which we fit a model of FR as a function of dry tissue weight and water temperature. We find that O. lurida has a FR at optimum temperature similar to previously established means across oyster species at 1 g dry tissue weight (DTW), but lower than many Crassostrea species. We also find that the allometric exponent relating FR to DTW in O. lurida is lower than the previously published mean across oyster species. Based on our derived filtration rates and historical data, we estimate the historic impact of filtration by O. lurida in five Pacific coast estuaries. We find that historic O. lurida populations did not have the capacity to filter the full volume of the estuary within the estuary residence time in any of the estuaries examined. This result is primarily driven by the low water temperatures and the short estuary residence times that typify the Pacific coast. We conclude that, unlike Crassostrea virginica Gmelin on the Atlantic and Gulf coasts, the Olympia oyster was not historically a dominant force in regulating seston concentrations at large scales in Pacific coast estuaries. Given the differences in the ecological role and habitat structure of these two oyster species, we recommend that analogies between them be drawn with caution. We discuss the implications of our results for developing restoration objectives.