Direct observations of the kinetics of migrating T-cells suggest active retention by endothelial cells with continual bidirectional migration.

The kinetics and regulatory mechanisms of T-cell migration through endothelium have not been fully defined. In experimental filter-based assays in vitro, transmigration of lymphocytes takes hours, compared to minutes in vivo. We cultured endothelial cell (EC) monolayers on filters, solid substrates or collagen gels, and treated them with tumour necrosis factor-α (TNF), interferon-γ (IFN), or both, prior to analysis of lymphocyte migration in the presence or absence of flow. Peripheral blood lymphocytes (PBL), CD4+ cells or CD8+ cells, took many hours to migrate through EC-filter constructs for all cytokine treatments. However, direct microscopic observations of EC-filters which had been mounted in a flow chamber showed that PBL crossed the endothelial monolayer in minutes and were highly motile in the subendothelial space. Migration through EC was also observed on clear plastic, with or without flow. After brief settling without flow, PBL and isolated CD3+ or CD4+ cells all crossed EC in minutes, but the numbers of migrated cells varied little with time. Close observation revealed that lymphocytes continuously migrated back and forth across endothelium. Under flow, migration kinetics and the proportions migrating back and forth were little altered. On collagen gels, PBL again crossed EC in minutes and migrated back and forth, but showed little penetration of the gel over hours.In contrast, neutrophils migrated efficiently through EC and into gels. These observations suggest a novel model for lymphoid migration, in which endothelial cells support migration but retain lymphocytes (as opposed to neutrophils), and additional signal(s) are required for onward migration

Rejuvenation of leukocyte trafficking in aged mice through PEPITEM intervention

Inflammageing leads to uncontrolled leukocyte trafficking in response to inflammatory insults. Here, we used a zymosan-induced peritonitis mouse model on inflammation to investigate the role of the PEPITEM pathway on leukocyte migration in ageing. We then analysed whether PEPITEM could modulate leukocyte migration in older adults. We observed a loss of functionality in the PEPITEM pathway, which normally controls leukocyte trafficking in response to inflammation, in older adults and aged mice and show that this can be rescued by supplementation with PEPITEM. Thus, leading to the exciting possibility that PEPITEM supplementation may represent a potential pre-habilitation geroprotective agent to rejuvenate immune functions

Analysis of the effects of stromal cells on the migration of lymphocytes into and through inflamed tissue using 3-D culture models

AbstractStromal cells may regulate the recruitment and behaviour of leukocytes during an inflammatory response, potentially through interaction with the endothelial cells (EC) and the leukocytes themselves. Here we describe new in vitro methodologies to characterise the effects of stromal cells on the migration of lymphocytes through endothelium and its underlying matrix. Three-dimensional tissue-like constructs were created in which EC were cultured above a stromal layer incorporating fibroblasts either as a monolayer on a porous filter or dispersed within a matrix of collagen type 1. A major advantage of these constructs is that they enable each step in leukocyte migration to be analysed in sequence (migration through EC and then stroma), as would occur in vivo. Migrated cells can also be retrieved from the constructs to identify which subsets traffic more effectively and how their functional responses evolve during migration. We found that culture of EC with dermal fibroblasts promoted lymphocyte transendothelial migration but not onward transit through matrix. A critical factor influencing the effect of fibroblasts on recruitment proved to be their proximity to the EC, with direct contact tending to disrupt migration. Comparison of the different approaches indicates that choice of an appropriate 3-D model enables the steps in lymphocyte entry into tissue to be studied in sequence, the regulatory mechanism to be dissected, and the effects of changes in stroma to be investigated

COMBREX: a project to accelerate the functional annotation of prokaryotic genomes

COMBREX (http://combrex.bu.edu) is a project to increase the speed of the functional annotation of new bacterial and archaeal genomes. It consists of a database of functional predictions produced by computational biologists and a mechanism for experimental biochemists to bid for the validation of those predictions. Small grants are available to support successful bids.National Institute of General Medical Sciences (U.S.) (Go grant 1RC2GM092602-01

Dementia as a disability: Implications for ethics, policy and practice. A Discussion Paper.

noPeople experience dementia in different ways, not just in terms of the type and severity of symptoms, but also in terms of how they react to and manage living with dementia. Increasingly, people with dementia are expressing a desire to get on with their everyday lives. They want to avoid being defined solely in relation to dementia and to continue to be considered as valued members of society. This is particularly important as the term dementia often has negative connotations. It is widely considered as a stigma. Neurological impairment may interfere with people’s ability to get on with their lives, as may differences in coping skills, financial resources, the emotional and psychological impact of dementia, and access to timely and good quality support. Reactions of relatives, friends and fellow citizens are also important, as well as society’s response to dementia. This was highlighted by Kitwood in the 1990s when he outlined what came to be known as the biopsychosocial model of dementia . There are also differences at the level of society, reflected in practices, attitudes and structures. These may, on the surface, seem fair or neutral (i.e. “that’s just the way it is”). In many cases, however, they reflect a lack of consideration and failure to act in a responsible, ethical and even legal way towards people with dementia... In this report, we focus on the possible implications for ethics, policy and practice of raising awareness about the potential of framing dementia as a potential disability

COMBREX: a project to accelerate the functional annotation of prokaryotic genomes

COMBREX (http://combrex.bu.edu) is a project to increase the speed of the functional annotation of new bacterial and archaeal genomes. It consists of a database of functional predictions produced by computational biologists and a mechanism for experimental biochemists to bid for the validation of those predictions. Small grants are available to support successful bids.National Institute of General Medical Sciences (U.S.) (Go grant 1RC2GM092602-01

Intracellular Serine Protease Inhibitor SERPINB4 Inhibits Granzyme M-Induced Cell Death

Granzyme-mediated cell death is the major pathway for cytotoxic lymphocytes to kill virus-infected and tumor cells. In humans, five different granzymes (i.e. GrA, GrB, GrH, GrK, and GrM) are known that all induce cell death. Expression of intracellular serine protease inhibitors (serpins) is one of the mechanisms by which tumor cells evade cytotoxic lymphocyte-mediated killing. Intracellular expression of SERPINB9 by tumor cells renders them resistant to GrB-induced apoptosis. In contrast to GrB, however, no physiological intracellular inhibitors are known for the other four human granzymes. In the present study, we show that SERPINB4 formed a typical serpin-protease SDS-stable complex with both recombinant and native human GrM. Mutation of the P2-P1-P1′ triplet in the SERPINB4 reactive center loop completely abolished complex formation with GrM and N-terminal sequencing revealed that GrM cleaves SERPINB4 after P1-Leu. SERPINB4 inhibited GrM activity with a stoichiometry of inhibition of 1.6 and an apparent second order rate constant of 1.3×104 M−1s−1. SERPINB4 abolished cleavage of the macromolecular GrM substrates α-tubulin and nucleophosmin. Overexpression of SERPINB4 in tumor cells inhibited recombinant GrM-induced as well as NK cell-mediated cell death and this inhibition depended on the reactive center loop of the serpin. As SERPINB4 is highly expressed by squamous cell carcinomas, our results may represent a novel mechanism by which these tumor cells evade cytotoxic lymphocyte-induced GrM-mediated cell death

Surface-initiated growth of copper using isonicotinic acid-functionalized aluminum oxide surfaces

Isonicotinate self-assembled monolayers (SAM) were prepared on alumina surfaces (A) using isonicotinic acid (iNA). These functionalized layers (iNA-A) were used for the seeded growth of copper films (Cu-iNA-A) by hydrazine hydrate-initiated electroless deposition. The films were characterized by scanning electron microscopy (SEM), electron-dispersive X-ray spectroscopy, atomic force microscopy, X-ray photoelectron spectroscopy, X-ray diffraction, and advancing contact angle measurements. The films are Cu0 but with surface oxidation, and show a faceted morphology, which is more textured (Rq = 460 ± 90 nm) compared to the SAM (Rq = 2.8 ± 0.5 nm). In contrast, growth of copper films by SnCl2/PdCl2 catalyzed electroless deposition, using formaldehyde (CH2O) as the reducing agent, shows a nodular morphology on top of a relatively smooth surface. No copper films are observed in the absence of the isonicotinate SAM. The binding of Cu2+ to the iNA is proposed to facilitate reduction to Cu0 and create the seed for subsequent growth. The films show good adhesion to the functionalized surface

Type-H endothelial cell protein Clec14a orchestrates osteoblast activity during trabecular bone formation and patterning

Type-H capillary endothelial cells control bone formation during embryogenesis and postnatal growth but few signalling mechanisms underpinning this influence have been characterised. Here, we identify a highly expressed type-H endothelial cell protein, Clec14a, and explore its role in coordinating osteoblast activity. Expression of Clec14a and its ligand, Mmrn2 are high in murine type-H endothelial cells but absent from osteoblasts. Clec14a−/− mice have premature condensation of the type-H vasculature and expanded distribution of osteoblasts and bone matrix, increased long-bone length and bone density indicative of accelerated skeletal development, and enhanced osteoblast maturation. Antibody-mediated blockade of the Clec14a-Mmrn2 interaction recapitulates the Clec14a−/− phenotype. Endothelial cell expression of Clec14a regulates osteoblast maturation and mineralisation activity during postnatal bone development in mice. This finding underscores the importance of type-H capillary control of osteoblast activity in bone formation and identifies a novel mechanism that mediates this vital cellular crosstalk