Endothelial chemokines destabilize L-selectin-mediated lymphocyte rolling without inducing selectin shedding

Chemokines presented on specialized endothelial surfaces rapidly up-regulate leukocyte integrin avidity and firm arrest through G(i)-protein signaling. Here we describe a novel, G-protein-independent, down-regulatory activity of apical endothelial chemokines in destabilizing L-selectin-mediated leukocyte rolling. Unexpectedly, this anti-adhesive chemokine suppression of rolling does not involve L-selectin shedding. Destabilization of rolling is induced only by immobilized chemokines juxtaposed to L-selectin ligands and is an energy-dependent process. Chemokines are found to interfere with a subsecond stabilization of selectin tethers necessary for persistent rolling. This is a first indication that endothelial chemokines can attenuate in situ L-selectin adhesion to endothelial ligands at subsecond contacts. This negative feedback mechanism may underlie the jerky nature of rolling mediated by L-selectin in vivo.     

Preferential Th1 immune response in invariant chain-deficient mice

MHC class II molecules associate with the invariant chain (Ii) molecule during biosynthesis. Ii facilitates the folding of class II molecules, interferes with their peptide association, and is involved in MHC class II transport. In this study, we have investigated the in vitro and in vivo immune response of Ii-deficient mice (Ii(-/-)). Our results have demonstrated that CD4(+) T cells from Ii(-/-) mice proliferate normally in vitro after in vivo immunization with protein Ags. However, cytokine secretion profiles of Ag-primed CD4(+) T cells from Ii(-/-) mice differ from CD4(+) T cells from wild-type mice. Whereas cells from wild-type mice secrete IFN-gamma and IL-4, cells from Ii(-/-) mice secrete mostly IFN-gamma. Moreover, Ii(-/-) mice exhibit a normal Th1 response in the delayed-type hypersensitivity and trinitrobenzene sulfonic acid colitis models; however, these mice lack an in vivo Th2 response, as demonstrated in the asthma model. Therefore, we suggest that defective Ag presentation in Ii(-/-) mice leads selectively to a Th1 effector response.     

Geometric algorithms for the analysis of 2D-electrophoresis gels.

In proteomics, two-dimensional gel electrophoresis (2-DE) is a separation technique for proteins. The resulting protein spots can be identified either by using picking robots and subsequent mass spectrometry or by visual cross inspection of a new gel image with an already analyzed master gel. Difficulties especially arise from inherent noise and irregular geometric distortions in 2-DE images. Aiming at the automated analysis of large series of 2-DE images, or at the even more difficult interlaboratory gel comparisons, the bottleneck is to solve the two most basic algorithmic problems with high quality: Identifying protein spots and computing a matching between two images. For the development of the analysis software CAROl at Freie Universit?t Berlin, we have reconsidered these two problems and obtained new solutions which rely on methods from computational geometry. Their novelties are: 1. Spot detection is also possible for complex regions formed by several "merged" (usually saturated) spots; 2. User-defined landmarks are not necessary for the matching. Furthermore, images for comparison are allowed to represent different parts of the entire protein pattern, which only partially "overlap." The implementation is done in a client server architecture to allow queries via the internet. We also discuss and point at related theoretical questions in computational geometry.     

Computational analysis of alternative splicing using EST tissue information

Expressed sequence tags (ESTs) from normal and tumor tissues have been deposited in public databases. These ESTs and all mRNA sequences were aligned with the human genome sequence using LEADS, Compugen's alternative splicing modeling platform. We developed a novel computational approach to analyze tissue information of aligned ESTs in order to identify cancer-specific alternative splicing and gene segments highly expressed in particular cancers. Several genes, including one encoding a possible pre-mRNA splicing factor, displayed cancer-specific alternative splicing. In addition, multiple candidate gene segments highly expressed in colon cancers were identified.     

Replicating by the clock

The eukaryotic genome is divided into well-defined DNA regions that are programmed to replicate at different times during S phase. Active genes are generally associated with early replication, whereas inactive genes replicate late. This expression pattern might be facilitated by the differential restructuring of chromatin at the time of replication in early or late S phase.     

TNF-alpha associated with extracellular matrix fibronectin provides a stop signal for chemotactically migrating T cells

The migration of T cells into extravascular sites of inflammation is regulated by information derived from the molecular structure of the invaded tissue and from chemokine and cytokine gradients in the context of the extracellular matrix (ECM). Although recent studies have highlighted the role of particular chemoattractants in leukocyte migration, to date little is known about how specific combinations of contextual signals control the migration of leukocytes and their localization at sites of inflammation. Here we studied the interplay between a pleiotropic cytokine, TNF-alpha, and two prototypic chemoattractants, RANTES and stromal cell-derived factor-1alpha (SDF-1alpha), on human CD45RO+ T cells migrating within an ECM-like context. For this purpose, we used a newly constructed three-dimensional gel system designed to follow, in real time, the migration of individual leukocytes along chemotactic gradients in vitro. We found that TNF-alpha, which binds the ECM protein fibronectin and lacks adhesion- and migration-promoting effects of its own, can act as a proadhesive cytokine on T cells exposed to RANTES and SDF-1alpha. Furthermore, fibronectin-complexed TNF-alpha provided anchorage signals to the T cells as they moved directionally along chemoattractive gradients. This effect of TNF-alpha required an intact TNF-alpha receptor II subtype on the migrating T cells. The anchoring effect of TNF-alpha appears to be specific; IL-2, an integrin-activating proadhesive cytokine, does not transmit stoppage signals to T cell migration induced by RANTES. Thus, TNF-alpha present in the ECM at sites of inflammation may function to anchor T cells recruited to these sites by chemotactic signals.     

Computerized analysis of chemotaxis at different stages of bacterial growth

Chemotactic behavior as a function of growth stage in an Escherichia coli strain commonly used for chemotaxis studies was characterized using computerized image analysis. The response and adaptation to saturating, step-like additions of the attractant L-aspartate were measured. Steady-state average tumbling frequency and adaptation time increased nearly twofold during logarithmic phase. In contrast, precision of adaptation, P, defined as the ratio between steady-state tumbling frequencies in the presence and absence of attractant, appeared to be constant throughout growth (P = 1.0 +/- 0.2). The variation of tumble duration over growth was consistent with a hydrodynamic mechanism for tumble termination.     

Predicting and controlling the reactivity of immune cell populations against cancer

Heterogeneous cell populations form an interconnected network that determine their collective output. One example of such a heterogeneous immune population is tumor‐infiltrating lymphocytes (TILs), whose output can be measured in terms of its reactivity against tumors. While the degree of reactivity varies considerably between different TILs, ranging from null to a potent response, the underlying network that governs the reactivity is poorly understood. Here, we asked whether one can predict and even control this reactivity. To address this we measured the subpopulation compositions of 91 TILs surgically removed from 27 metastatic melanoma patients. Despite the large number of subpopulations compositions, we were able to computationally extract a simple set of subpopulation‐based rules that accurately predict the degree of reactivity. This raised the conjecture of whether one could control reactivity of TILs by manipulating their subpopulation composition. Remarkably, by rationally enriching and depleting selected subsets of subpopulations, we were able to restore anti‐tumor reactivity to nonreactive TILs. Altogether, this work describes a general framework for predicting and controlling the output of a cell mixture.     

Multi-node graphs: a framework for multiplexed biological assays.

Multiplex polymerase chain reaction (PCR) is an extension of the standard PCR protocol in which primers for multiple DNA loci are pooled together within a single reaction tube, enabling simultaneous sequence amplification, thus reducing costs and saving time. Potential cost saving and throughput improvements directly depend on the level of multiplexing achieved. Designing reliable and highly multiplexed assays is challenging because primers that are pooled together in a single reaction tube may cross-hybridize, though this can be addressed either by modifying the choice of primers for one or more amplicons, or by altering the way in which DNA loci are partitioned into separate reaction tubes. In this paper, we introduce a new graph formalism called a multi-node graph, and describe its application to the analysis of multiplex PCR scalability. We show, using random multi-node graphs that the scalability of multiplex PCR is constrained by a phase transition, suggesting fundamental limits on efforts to improve the cost-effectiveness and throughput of standard multiplex PCR assays. In particular, we show that when the multiplexing level of the reaction tubes is roughly theta(log (sn)) (where s is the number of primer pair candidates per locus and n is the number of loci to be amplified), then with very high probability we can 'cover' all loci with a valid assignment to one of the tubes in the assay. However, when the multiplexing level of the tube exceeds these bounds, there is no possible cover and moreover the size of the cover drops dramatically. Simulations using a simple greedy algorithm on real DNA data also confirm the presence of this phase transition. Our theoretical results suggest, however, that the resulting phase transition is a fundamental characteristic of the problem, implying intrinsic limits on the development of future assay design algorithms.