SUMMARY European and North American strains of the parasite Toxoplasma gondii belong to three distinct clonal lineages, type I, II and III, which differ in virulence. Understanding the basis of Toxoplasma strain differences and how secreted effectors work to achieve chronic infection is a major goal of current research. Here we show that type I and III infected macrophages, a cell type required for host immunity to Toxoplasma, are alternatively activated, while type II infected macrophages are classically activated. The Toxoplasma rhoptry kinase ROP16, which activates STAT6, is responsible for alternative activation. The Toxoplasma dense granule protein GRA15, which activates NF-κB, promotes classical activation by type II parasites. These effectors antagonistically regulate many of the same genes, and mice infected with type II parasites expressing type I ROP16 are protected against Toxoplasma-induced ileitis. Thus, polymorphisms in determinants that modulate macrophage activation influence the ability of Toxoplasma to establish a chronic infection.

The UCSF COMET Consortium, Michael Matthay, David J. E rl e , P re scot t G. Woodruff, Charles Langelier, Kirsten K an ge la ri s, C ar ol yn M.

The extracellular matrix (ECM) is a complex assembly of structural proteins that provides physical support and biochemical signaling to cells in tissues. The mechanical properties of the ECM have been found to play a key role in regulating cell behaviors such as differentiation and malignancy. Gels formed from ECM protein biopolymers such as collagen or fibrin are commonly used for 3D cell culture models of tissue. One of the most striking features of these gels is that they exhibit nonlinear elasticity, undergoing strain stiffening. However, these gels are also viscoelastic and exhibit stress relaxation, with the resistance of the gel to a deformation relaxing over time. Recent studies have suggested that cells sense and respond to both nonlinear elasticity and viscoelasticity of ECM, yet little is known about the connection between nonlinear elasticity and viscoelasticity. Here, we report that, as strain is increased, not only do biopolymer gels stiffen but they also exhibit faster stress relaxation, reducing the timescale over which elastic energy is dissipated. This effect is not universal to all biological gels and is mediated through weak cross-links. Mechanistically, computational modeling and atomic force microscopy (AFM) indicate that strain-enhanced stress relaxation of collagen gels arises from force-dependent unbinding of weak bonds between collagen fibers. The broader effect of strain-enhanced stress relaxation is to rapidly diminish strain stiffening over time. These results reveal the interplay between nonlinear elasticity and viscoelasticity in collagen gels, and highlight the complexity of the ECM mechanics that are likely sensed through cellular mechanotransduction.collagen mechanics | viscoelasticity | force-dependent unbinding | biopolymers | stress relaxation T he composition and architecture of ECM is heterogeneous and varies with tissue type and location. One particularly important ECM protein is type Ι collagen, which is the most abundant ECM component and primarily determines the mechanics of connective tissue (1). Type 1 collagen self-assembles into fibers, and these fibers can form networks in vitro. Studies investigating the mechanical properties of collagen networks have revealed that these networks are nonlinearly elastic and exhibit strain stiffening, or an increase in the elasticity as the strain on the network is enhanced (1-3). This nonlinear elasticity is also a characteristic feature of fibrin gels, which serve as the major component of blood clots, as well as in reconstituted networks of intermediate filaments and cytoskeletal actin networks (2, 4-7). These networks are all composed of semiflexible polymers or fibers, which are relatively rigid, so that the tangent to the contour of the polymer is correlated over long lengths, yet undergo substantial bending fluctuations due to thermal energy. Semiflexible polymers or fibers form networks at low volume fractions (8). Strain stiffening in these networks is thought to arise from either the entropic elasticity of single polymers...

The T cell receptor requires force for triggering. Here, Hu and Butte show that T cells generate pushing and pulling forces against an antigen-coated AFM cantilever in an actin-dependent fashion. Exogenous, oscillating forces delivered by the cantilever rescued T cell receptor signaling in the absence of an intact F-actin cytoskeleton. These findings highlight the importance of mechanical forces in T cell activation.

In the human genome, the erythroidspecfic hypersensitive site HS2 enhancer regulates the trsiption of the downstream 1-like globin genes 10-50 kilobses away. The mechanism of HS2 eancr fuction Is not known. The present study employs RNA protection assays to analyze the transcriptional status of the HS2 enancer in trneced recombinant chloramphenicol acetltrnsferase (CAT) pasmids. In erythroid K562 cells in which the HS2 enhancer is active, the HS2 sequence directs the synthesis of long enhancer transcripts that are initiated apparently from within the enhancer and elongated through the intervening DNA into the cis-linked CAT gene. In nonerythroid HL-60 cells in which the HS2 enhancer is inactive, long enhancer tra rpt are not detectable. Splitting the HS2 encer between two tandem Apl sites abolishes the synthesis of a group of log enhancer transcripts and results in loss of enhancer function and transcriptional silencing ofthe cis-linked CAT gene. In directing the synthesis ofRNA through the intervening DNA and the gene by a tracking and transcription mechan , the HS2 enhaner may (i) open up the chromatin structure of a gene domain and (it) deliver enhancer binding proteins to the promoter sequence where they may stimulate the transcription of the gene at the cap site.In seeking to identify the cis regulatory elements of the human (-like globin genes ( this laboratory (1) and others (2, 3) have mapped four erythroid-specific and developmentally stable DNase I-hypersensitive sites HS1, -2, -3, and -4 in the locus control region (LCR) between 50 and 70 kilobases (kb) upstream of the (3-globin gene. HS2 at -11 kb 5' of the embryonic E-and thus at -54 kb 5' of the ,B-globin gene has been shown to possess an erythroidspecific and developmentally stable enhancer function (4-6). It is capable of stimulating the transcription of embryonic E-, fetal -, and adult (B-globin genes in erythroid cells (7-10). In y6f3thalassemia, the deletion of HS2 and more upstream DNA is associated with transcriptional silencing of the far downstream f3-globin gene (11-13) and possibly of the whole (-like globin gene domain.How the HS2 enhancer cooperates with distant globin promoter sequences to stimulate the E-, y-, and (-globin genes at the respective developmental stage is not clear. Looping (14) and tracking (15) mechanisms have been proposed to explain how a distant enhancer may communicate with the promoter of a cis-linked gene (14,16,17). In the present study, the detection of HS2 enhancer transcripts in erythroid cells suggests that the tandem Apl sites and other sequence motifs in the enhancer may provide entry sites for transcription factors associated with the transcriptional machinery that track the DNA and synthesize long strands of RNA through the intervening DNA into the cis-linked gene.The possible biological significance of such long transcripts in enhancer function will be discussed. MATERIALS AND METHODSConstruction ofRecombinant Constructs. HS2-eP-CAT and EP-CAT have been described (5). 5'-HS2-eP-CAT was made by ...